Cell杂志（6月15日）文章完全版

　　

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Article

Nef-Mediated Suppression of T Cell Activation Was Lost in a Lentiviral Lineage that Gave Rise to HIV-1

Michael Schindler,¹ Jan Münch,¹ Olaf Kutsch,² Hui Li,² Mario L. Santiago,² Frederic Bibollet-Ruche,² Michaela C. Müller-Trutwin,³ Francis J. Novembre,⁴ Martine Peeters,⁵ Valerie Courgnaud,⁵ Elizabeth Bailes,⁶ Pierre Roques,⁷ Donald L. Sodora,⁸ Guido Silvestri,^4,9 Paul M. Sharp,⁶ Beatrice H. Hahn,² and Frank Kirchhoff^1,

¹ Department of Virology, University of Ulm, 89081 Ulm, Germany
² Departments of Medicine and Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
³ Unité de Biologie des Rétrovirus, Institut Pasteur, 75015 Paris, France
⁴ Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30329, USA
⁵ Laboratoire Retrovirus, UMR145, Institut de Recherche pour le Developpement and Department of International Health, University of Montpellier, 34032 Montpellier, France
⁶ Institute of Genetics, University of Nottingham, Queens Medical Centre, NG7 2UH, Nottingham, UK
⁷ Département de Virologie, CIRMF, BP769 Franceville, Gabon
⁸ Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
⁹ Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

High-level immune activation and T cell apoptosis represent a hallmark of HIV-1 infection that is absent from nonpathogenic SIV infections in natural primate hosts. The mechanisms causing these varying levels of immune activation are not understood. Here, we report that nef alleles from the great majority of primate lentiviruses, including HIV-2, downmodulate TCR-CD3 from infected T cells, thereby blocking their responsiveness to activation. In contrast, nef alleles from HIV-1 and a subset of closely related SIVs fail to downregulate TCR-CD3 and to inhibit cell death. Thus, Nef-mediated suppression of T cell activation is a fundamental property of primate lentiviruses that likely evolved to maintain viral persistence in the context of an intact host immune system. This function was lost during viral evolution in a lineage that gave rise to HIV-1 and may have predisposed the simian precursor of HIV-1 for greater pathogenicity in humans.

Article

rRNA Promoter Regulation by Nonoptimal Binding of σ Region 1.2: An Additional Recognition Element for RNA Polymerase

Shanil P. Haugen,¹ Melanie B. Berkmen,^1,2 Wilma Ross,¹ Tamas Gaal,¹ Christopher Ward,^1,3 and Richard L. Gourse^1,

¹ Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA

Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that σ subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the −10 element (within the “discriminator” region). By making a nonoptimal σ1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.

Article  （cover story）

Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules

Kristina M. Herbert,¹ Arthur La Porta,² Becky J. Wong,² Rachel A. Mooney,³ Keir C. Neuman,^2,5 Robert Landick,³ and Steven M. Block^2,4,

¹ Biophysics Program, Stanford University, Stanford, CA 94305, USA
² Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
³ Department of Bacteriology, University of Wisconsin—Madison, Madison, WI 53706, USA
⁴ Department of Applied Physics, Stanford University, Stanford, CA 94305, USA

Summary

Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these “ubiquitous” pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

Article

Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies

Ujwal Sheth¹ and Roy Parker^1,2,

¹ Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
² Howard Hughes Medical Institute, University of Arizona, Tucson, AZ 85721, USA

Summary

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.

Article

Relief of microRNA-Mediated Translational Repression in Human Cells Subjected to Stress

Suvendra N. Bhattacharyya,¹ Regula Habermacher,¹ Ursula Martine,² Ellen I. Closs,² and Witold Filipowicz^1,

¹ Friedrich Miescher Institute for Biomedical Research, P.O. Box 2543, 4002 Basel, Switzerland
² Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany

Summary

In metazoans, most microRNAs imperfectly base-pair with the 3′ untranslated region (3′UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3′UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3′UTR of CAT-1 mRNA. We propose that proteins interacting with the 3′UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Article

In Vitro Reconstitution of Eukaryotic Translation Reveals Cooperativity between Release Factors eRF1 and eRF3

Elena Z. Alkalaeva,^1,2,4 Andrey V. Pisarev,^1,4 Lyudmila Y. Frolova,² Lev L. Kisselev,^2, and Tatyana V. Pestova^1,3,

¹ Department of Microbiology and Immunology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA
² Engelhardt Institute of Molecular Biology, The Russian Academy of Sciences, 119991 Moscow, Russia
³ A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia

Summary

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.

Article

An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor

Xuewu Zhang,¹ Jodi Gureasko,¹ Kui Shen,^3,4 Philip A. Cole,³ and John Kuriyan^1,2,

¹ Department of Molecular and Cell Biology and Department of Chemistry and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA
² Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
³ Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Summary

The mechanism by which the epidermal growth factor receptor (EGFR) is activated upon dimerization has eluded definition. We find that the EGFR kinase domain can be activated by increasing its local concentration or by mutating a leucine (L834R) in the activation loop, the phosphorylation of which is not required for activation. This suggests that the kinase domain is intrinsically autoinhibited, and an intermolecular interaction promotes its activation. Using further mutational analysis and crystallography we demonstrate that the autoinhibited conformation of the EGFR kinase domain resembles that of Src and cyclin-dependent kinases (CDKs). EGFR activation results from the formation of an asymmetric dimer in which the C-terminal lobe of one kinase domain plays a role analogous to that of cyclin in activated CDK/cyclin complexes. The CDK/cyclin-like complex formed by two kinase domains thus explains the activation of EGFR-family receptors by homo- or heterodimerization.

Article

EphB Receptors Coordinate Migration and Proliferation in the Intestinal Stem Cell Niche

Johan Holmberg,^1,3 Maria Genander,^1,3 Michael M. Halford,² Cecilia Annerén,¹ Mariann Sondell,¹ Michael J. Chumley,² Robert E. Silvany,² Mark Henkemeyer,² and Jonas Frisén^1,

¹ Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden
² Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

Summary

More than 10¹⁰ cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.

Article

C. elegans SIR-2.1 Interacts with 14-3-3 Proteins to Activate DAF-16 and Extend Life Span

Ala Berdichevsky,^1,2 Mohan Viswanathan,² H. Robert Horvitz,^1,2 and Leonard Guarente^2,

¹ Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
² Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Summary

The longevity of Caenorhabditis elegans is promoted by extra copies of the sir-2.1 gene in a manner dependent on the forkhead transcription factor DAF-16. We identify two C. elegans 14-3-3 proteins as SIR-2.1 binding partners and show that 14-3-3 genes are required for the life-span extension conferred by extra copies of sir-2.1. 14-3-3 proteins are also required for SIR-2.1-induced transcriptional activation of DAF-16 and stress resistance. Following heat stress, SIR-2.1 can bind DAF-16 in a 14-3-3-dependent manner. By contrast, low insulin-like signaling does not promote SIR-2.1/DAF-16 interaction, and sir-2.1 and the 14-3-3 genes are not required for the regulation of life span by the insulin-like signaling pathway. We propose the existence of a stress-dependent pathway in which SIR-2.1 and 14-3-3 act in parallel to the insulin-like pathway to activate DAF-16 and extend life span.

Article

Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration Due to Mutant Huntingtin

Rona K. Graham,^1,3 Yu Deng,^1,3 Elizabeth J. Slow,^1,3 Brendan Haigh,^1,3 Nagat Bissada,^1,3 Ge Lu,^1,3 Jacqueline Pearson,^1,3 Jacqueline Shehadeh,² Lisa Bertram,^1,3 Zoe Murphy,^1,3 Simon C. Warby,^1,3 Crystal N. Doty,^1,3 Sophie Roy,⁴ Cheryl L. Wellington,³ Blair R. Leavitt,^1,3 Lynn A. Raymond,² Donald W. Nicholson,⁴ and Michael R. Hayden^1,3,

¹ Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada
² Department of Psychiatry, University of British Columbia, Vancouver, BC V5Z 4H4, Canada
³ Child and Family Research Institute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada
⁴ Merck Research Laboratories, Whitehouse Station, NJ 08889, USA

Summary

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Resource

A Gene-Centered C. elegans Protein-DNA Interaction Network

Bart Deplancke,¹ Arnab Mukhopadhyay,^1,6 Wanyuan Ao,^2,5,6 Ahmed M. Elewa,¹ Christian A. Grove,¹ Natalia J. Martinez,¹ Reynaldo Sequerra,³ Lynn Doucette-Stamm,³ John S. Reece-Hoyes,⁴ Ian A. Hope,⁴ Heidi A. Tissenbaum,¹ Susan E. Mango,² and Albertha J.M. Walhout^1,

¹ Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
² Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112, USA
³ Agencourt Bioscience Corporation, Beverly MA 01915, USA
⁴ Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, School of Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, United Kingdom

Summary

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using “TF-centered” methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used “gene-centered” high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for ∼10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.