Showing papers by "Howard Hughes Medical Institute published in 2005"
TL;DR: A new, bead-based flow cytometric miRNA expression profiling method is used to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers, and finds the miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours.
Abstract: Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.
9,470 citations
TL;DR: Insight is provided into the transcriptional regulation of stem cells and how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal and how they collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops.
4,447 citations
TL;DR: It is shown that a novel class of chemically engineered oligonucleotides, termed ‘antagomirs’, are efficient and specific silencers of endogenous miRNA levels in mice and may represent a therapeutic strategy for silencing miRNAs in disease.
Abstract: MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.
4,045 citations
TL;DR: Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation.
Abstract: Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.
3,279 citations
TL;DR: A requirement for VEGFR1+ haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells is demonstrated.
Abstract: The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.
2,923 citations
TL;DR: High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations.
2,898 citations
TL;DR: Analysis of Foxp3 expression during thymic development suggests that this mechanism is not hard-wired but is dependent on TCR/MHC ligand interactions, and it is shown that expression ofFoxp3 is highly restricted to the subset alphabeta of T cells and, irrespective of CD25 expression, correlates with suppressor activity.
2,248 citations
TL;DR: Estimation of transcripts encoding selected hypothalamic peptides associated with energy balance was attenuated in the Clock mutant mice, suggesting that the circadian clock gene network plays an important role in mammalian energy balance.
Abstract: The CLOCK transcription factor is a key component of the molecular circadian clock within pacemaker neurons of the hypothalamic suprachiasmatic nucleus. We found that homozygous Clock mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese, and develop a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis, hyperglycemia, and hypoinsulinemia. Expression of transcripts encoding selected hypothalamic peptides associated with energy balance was attenuated in the Clock mutant mice. These results suggest that the circadian clock gene network plays an important role in mammalian energy balance.
2,241 citations
TL;DR: In this paper, the authors reported the crystal structure of a mammalian voltage-dependent potassium ion (K+) channel, Kv1.2, which is a member of the Shaker K+ channel family.
Abstract: Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.
2,130 citations
TL;DR: S-nitrosylation conveys a large part of the ubiquitous influence of nitric oxide on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.
Abstract: S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.
2,006 citations
TL;DR: The atomic structure of the cross-β spine illuminates the stability of amyloid fibrils, their self-seeding characteristic and their tendency to form polymorphic structures.
Abstract: Numerous soluble proteins convert to insoluble amyloid-like fibrils that have common properties. Amyloid fibrils are associated with fatal diseases such as Alzheimer's, and amyloid-like fibrils can be formed in vitro. For the yeast protein Sup35, conversion to amyloid-like fibrils is associated with a transmissible infection akin to that caused by mammalian prions. A seven-residue peptide segment from Sup35 forms amyloid-like fibrils and closely related microcrystals, from which we have determined the atomic structure of the cross-β spine. It is a double β-sheet, with each sheet formed from parallel segments stacked in register. Side chains protruding from the two sheets form a dry, tightly self-complementing steric zipper, bonding the sheets. Within each sheet, every segment is bound to its two neighbouring segments through stacks of both backbone and side-chain hydrogen bonds. The structure illuminates the stability of amyloid fibrils, their self-seeding characteristic and their tendency to form polymorphic structures.
TL;DR: The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
Abstract: The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resolution of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The atomic details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
Research Institute of Molecular Pathology1, Ludwig Maximilian University of Munich2, University of Colorado Boulder3, University of Cambridge4, Centro Nacional de Investigaciones Cardiovasculares5, Autonomous University of Madrid6, University of Navarra7, University of Barcelona8, Howard Hughes Medical Institute9
TL;DR: It is found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4 early and accumulated during the tumorigenic process, which is a common hallmark of human tumor cells.
Abstract: CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.
TL;DR: Another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways is indicated, which regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.
Abstract: The transmission of extracellular signals to the interior of the cell is a function of plasma membrane receptors, of which the seven transmembrane receptor family is by far the largest and most versatile. Classically, these receptors stimulate heterotrimeric G proteins, which control rates of generation of diffusible second messengers and entry of ions at the plasma membrane. Recent evidence, however, indicates another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways. They direct the recruitment, activation, and scaffolding of cytoplasmic signaling complexes via two multifunctional adaptor and transducer molecules, beta-arrestins 1 and 2. This mechanism regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.
TL;DR: It is shown that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability and is regulated by AMP-activated protein kinase (AMPK), acell-cycle arrest that occurs despite continued amino acid availability and active mTOR.
TL;DR: Methylation patterns at orthologous loci are strongly conserved between human and mouse even though many methylated sites do not show sequence conservation notably higher than background, which suggests that the DNA elements that direct the methylation represent only a small fraction of the region or lie at some distance from the site.
TL;DR: It is demonstrated that mitochondria function as O(2) sensors and signal hypoxic Hif-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.
TL;DR: Synthetic lethality provides a conceptual framework for the development of cancer-specific cytotoxic agents and has not been exploited in the past because there were no robust methods for systematically identifying synthetic lethal genes.
Abstract: Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells. Synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents. This paradigm has not been exploited in the past because there were no robust methods for systematically identifying synthetic lethal genes. This is changing as a result of the increased availability of chemical and genetic tools for perturbing gene function in somatic cells.
TL;DR: By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.
Abstract: The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.
TL;DR: The conversion of truly naive CD4+ T cells into suppressor cells expressing Foxp3 is reported by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp 3 expression at the level of single cells, showing that conversion was achieved by minute antigen doses with suboptimal dendrite cell activation.
Abstract: Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4+ T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-β or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.
TL;DR: It is demonstrated that Argonaute proteins — the signature components of the RNA interference (RNAi) effector complex, RISC — localize to mammalian P-bodies and suggested that translation repression by RISC delivers mRNAs to P- bodies, either as a cause or as a consequence of inhibiting protein synthesis.
Abstract: Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.
TL;DR: Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance is described, with overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios.
TL;DR: The preclinical and clinical development of imatinib for the therapy of CML, resistance and strategies that may help to eliminate resistant or residual leukemia are reviewed.
TL;DR: Patients with advanced colorectal cancers consistently contained mutant adenomatous polyposis coli (APC) DNA molecules in their plasma, which has implications for the mechanisms through which tumor DNA is released into the circulation and for diagnostic tests based on this phenomenon.
Abstract: The early detection of cancers through analysis of circulating DNA could have a substantial impact on morbidity and mortality. To achieve this goal, it is essential to determine the number of mutant molecules present in the circulation of cancer patients and to develop methods that are sufficiently sensitive to detect these mutations. Using a modified version of a recently developed assay for this purpose, we found that patients with advanced colorectal cancers consistently contained mutant adenomatous polyposis coli (APC) DNA molecules in their plasma. The median number of APC DNA fragments in such patients was 47,800 per ml of plasma, of which 8% were mutant. Mutant APC molecules were also detected in >60% of patients with early, presumably curable colorectal cancers, at levels ranging from 0.01% to 1.7% of the total APC molecules. These results have implications for the mechanisms through which tumor DNA is released into the circulation and for diagnostic tests based on this phenomenon.
TL;DR: In 6-month-old mice, spines turn over more slowly in visual compared to somatosensory cortex, possibly reflecting differences in the capacity for experience-dependent plasticity in these brain regions.
TL;DR: It is shown that the intrinsic plasticity of the protein is a key characteristic of catalysis, and the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergy pressure between structure and dynamics.
Abstract: A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
TL;DR: It is reported that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for allImatinib-resistant mutants tested, with the exception of T315I.
Abstract: Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.
TL;DR: The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues.
Abstract: Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.
TL;DR: Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption and gave insight into hemochromatosis.
TL;DR: A comprehensive understanding of eukaryotic gene expression requires an appreciation for how the lives of mRNAs are influenced by a wide array of diverse regulatory mechanisms.
Abstract: Recent work indicates that the posttranscriptional control of eukaryotic gene expression is much more elaborate and extensive than previously thought, with essentially every step of messenger RNA (mRNA) metabolism being subject to regulation in an mRNA-specific manner. Thus, a comprehensive understanding of eukaryotic gene expression requires an appreciation for how the lives of mRNAs are influenced by a wide array of diverse regulatory mechanisms.