DSpace at MIT
This site is a university repository providing access to the publication output of the institution. Registered users can set up email alerts to notify them of newly added relevant content. A certain level of encryption and security is embedded in the site which may cause some users accessibility problems.
MIT Open Access Articles
Mostrando recursos 41 - 60 de 11,227
Identification of High Affinity Polo-like Kinase 1 (Plk1) Polo-box Domain Binding Peptides Using Oxime-Based Diversification - Liu, Fa; Park, Jung-Eun; Qian, Wen-Jian; Scharow, Andrej; Berg, Thorsten; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.; Lim, Daniel Cham-Chin
In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation...
Abrogation of antibody-induced arthritis in mice by a self-activating viridin prodrug and association with impaired neutrophil and endothelial cell function - Stangenberg, Lars; Ellson, Chris; Cortez-Retamozo, Virna; Ortiz-Lopez, Adriana; Yuan, Hushan; Blois, Joseph; Smith, Ralph A.; Yaffe, Michael B.; Weissleder, Ralph; Benoist, Christophe; Mathis, Diane; Josephson, Lee; Mahmood, Umar
To test a novel self-activating viridin (SAV) prodrug that slowly releases wortmannin, a potent phosphoinositide 3-kinase inhibitor, in a model of antibody-mediated inflammatory arthritis.
The SAV prodrug was administered to K/BxN mice or to C57BL/6 (B6) mice that had been injected with K/BxN serum. Ankle thickness was measured, and histologic changes were scored after a 10-day disease course (serum-transfer arthritis). Protease activity was measured by a near-infrared imaging approach using a cleavable cathepsin–selective probe. Further near-infrared imaging techniques were used to analyze early changes in vascular permeability after serum injection, as well as neutrophil–endothelial cell interactions. Neutrophil functions were assessed using...
Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation - Davies, Bryan William; Jacob, Asha I.; Koehrer, Caroline; Rajbhandary, Uttam L.; Walker, Graham C.
Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report evidence of a previously unrecognized ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a ΔybeY mutant at 45°C, making YbeY the only endoribonuclease to be implicated...
NMR Structure and Dynamics of the C-Terminal Domain from Human Rev1 and Its Complex with Rev1 Interacting Region of DNA Polymerase η - Pozhidaeva, Alexandra; Pustovalova, Yulia; Bezsonova, Irina; Walker, Graham C.; Korzhnev, Dmitry M.; D'Souza, Sanjay Victor
Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer–template. Rev1...
Oxidation of the Guanine Nucleotide Pool Underlies Cell Death by Bactericidal Antibiotics - Foti, James J.; Devadoss, Babho; Walker, Graham C.; Winkler, Jonathan A.; Collins, James J.
A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions,...
Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli - Cohen, Susan E.; Lewis, Cindi A.; Walker, Graham C.; Mooney, Rachel A.; Kohanski, Michael A.; Collins, James J.; Landick, Robert
We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression...
PAK Inactivation Impairs Social Recognition in 3xTg-AD Mice without Increasing Brain Deposition of Tau and Aβ - Tonegawa, Susumu; Arsenault, Dany; Dal-Pan, Alexandre; Tremblay, Cyntia; Bennett, David A.; Guitton, Matthieu J.; De Koninck, Yves; Calon, Frederic
Defects in p21-activated kinase (PAK) are suspected to play a role in cognitive symptoms of Alzheimer's disease (AD). Dysfunction in PAK leads to cofilin activation, drebrin displacement from its actin-binding site, actin depolymerization/severing, and, ultimately, defects in spine dynamics and cognitive impairment in mice. To determine the role of PAK in AD, we first quantified PAK by immunoblotting in homogenates from the parietal neocortex of subjects with a clinical diagnosis of no cognitive impairment (n = 12), mild cognitive impairment (n = 12), or AD (n = 12). A loss of total PAK, detected in the cortex of AD patients...
Unified Model for Contact Angle Hysteresis on Heterogeneous and Superhydrophobic Surfaces - Raj, Rishi; Enright, Ryan; Zhu, Yangying; Wang, Evelyn N.; Adera, Solomon E.
Understanding the complexities associated with contact line dynamics on chemically heterogeneous and superhydrophobic surfaces is important for a wide variety of engineering problems. Despite significant efforts to capture the behavior of a droplet on these surfaces over the past few decades, modeling of the complex dynamics at the three-phase contact line is needed. In this work, we demonstrate that contact line distortion on heterogeneous and superhydrophobic surfaces is the key aspect that needs to be accounted for in the dynamic droplet models. Contact line distortions were visualized and modeled using a thermodynamic approach to develop a unified model for contact...
MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma - Sabatini, David M.; Wood, Kris C.; Konieczkowski, David J.; Johannessen, Cory M.; Boehm, Jesse S.; Tamayo, Pablo; Botvinnik, Olga B.; Mesirov, Jill P.; Hahn, William C.; Root, David E.; Garraway, Levi A.
Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but owing to technical limitations they have been restricted for use with a narrow range of cell lines and short-term assays. Here, we describe MicroSCALE (Microarrays of Spatially Confined Adhesive Lentiviral Features), a cell microarray–based platform that enables application of this technology to a wide range of cell types and longer-term assays. We used MicroSCALE to uncover kinases that when overexpressed partially desensitized B-RAF[superscript V600E]–mutant melanoma cells to inhibitors of the mitogen-activated protein kinase kinase kinase (MAPKKK) RAF, the MAPKKs MEK1 and...
Unbiased reconstruction of a mammalian transcriptional network mediating the differential response to pathogens - Amit, Ido; Guttman, Mitchell; Leite, Ana Paula; Regev, Aviv; Garber, Manuel; Chevrier, Nicolas; Donner, Yoni; Eisenhaure, Thomas; Grenier, Jennifer K.; Li, Weibo; Zuk, Or; Schubert, Lisa A.; Birditt, Brian; Shay, Tal; Goren, Alon; Zhang, Xiaolan; Smith, Zachary; Deering, Raquel; McDonald, Rebecca C.; Cabili, Moran; Bernstein, Bradley E.; Rinn, John L.; Meissner, Alex; Root, David E.; Hacohen, Nir
Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a...
DHODH modulates transcriptional elongation in the neural crest and melanoma - White, Richard Mark; Cech, Jennifer; Ratanasirintrawoot, Sutheera; Lin, Charles Y.; Rahl, Peter B.; Burke, Christopher J.; Langdon, Erin; Tomlinson, Matthew L.; Mosher, Jack; Kaufman, Charles; Chen, Frank; Long, Hannah K.; Kramer, Martin; Datta, Sumon; Neuberg, Donna; Granter, Scott; Young, Richard A.; Morrison, Sean; Wheeler, Grant N.; Zon, Leonard I.
Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma1. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for...
Control of the Embryonic Stem Cell State - Young, Richard A.; Young, Richard A.
Embryonic stem cells and induced pluripotent stem cells hold great promise for regenerative medicine. These cells can be propagated in culture in an undifferentiated state but can be induced to differentiate into specialized cell types. Moreover, these cells provide a powerful model system for studies of cellular identity and early mammalian development. Recent studies have provided insights into the transcriptional control of embryonic stem cell state, including the regulatory circuitry underlying pluripotency. These studies have, as a consequence, uncovered fundamental mechanisms that control mammalian gene expression, connect gene expression to chromosome structure, and contribute to human disease.
DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization - Reinhardt, H. Christian; Hasskamp, Pia; Schmedding, Ingolf; Morandell, Sandra; van Vugt, Marcel A.T.M.; Wang, XiaoZhe; Linding, Rune; Ong, Shao-En; Weaver, David; Carr, Steven A.; Yaffe, Michael B.
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G2/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where...
Systemic Elevation of PTEN Induces a Tumor-Suppressive Metabolic State - Garcia-Cao, Isabel; Song, Min Sup; Hobbs, Robin M.; Laurent, Gaelle; Giorgi, Carlotta; de Boer, Vincent C.J.; Anastasiou, Dimitrios; Ito, Keisuke; Sasaki, Atsuo T.; Rameh, Lucia; Carracedo, Arkaitz; Vander Heiden, Matthew G.; Cantley, Lewis C.; Pinton, Paolo; Haigis, Marcia C.; Pandolfi, Pier Paolo; Vander Heiden, Matthew G.
Decremental loss of PTEN results in cancer susceptibility and tumor progression. PTEN elevation might therefore be an attractive option for cancer prevention and therapy. We have generated several transgenic mouse lines with PTEN expression elevated to varying levels by taking advantage of bacterial artificial chromosome (BAC)-mediated transgenesis. The “Super-PTEN” mutants are viable and show reduced body size due to decreased cell number, with no effect on cell size. Unexpectedly, PTEN elevation at the organism level results in healthy metabolism characterized by increased energy expenditure and reduced body fat accumulation. Cells derived from these mice show reduced glucose and glutamine uptake...
Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells - Vander Heiden, Matthew G.; Locasale, Jason W.; Swanson, Kenneth D.; Sharfi, Hadar; Heffron, Greg J.; Amador-Noguez, Daniel; Christofk, Heather R.; Wagner, Gerhard; Rabinowitz, Joshua D.; Asara, John M.; Cantley, Lewis C.
Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the...
Identification and optogenetic manipulation of memory engrams in the hippocampus - Ramirez Moreno, Steve; Tonegawa, Susumu; Liu, Xu
With the accumulation of our knowledge about how memories are formed, consolidated, retrieved, and updated, neuroscience is now reaching a point where discrete memories can be identified and manipulated at rapid timescales. Here, we start with historical studies that lead to the modern memory engram theory. Then, we will review recent advances in memory engram research that combine transgenic and optogenetic approaches to reveal the underlying neuronal substrates sufficient for activating mnemonic processes. We will focus on three concepts: (1) isolating memory engrams at the level of single cells to tag them for subsequent manipulation; (2) testing the sufficiency of...
Distinct preplay of multiple novel spatial experiences in the rat - Dragoi, George; Tonegawa, Susumu
The activity of ensembles of hippocampal place cells represents a hallmark of an animal’s spatial experience. The neuronal mechanisms that enable the rapid expression of novel place cell sequences are not entirely understood. Here we report that during sleep or rest, distinct sets of hippocampal temporal sequences in the rat preplay multiple corresponding novel spatial experiences with high specificity. These findings suggest that the place cell sequence of a novel spatial experience is determined, in part, by an online selection of a subset of cellular firing sequences from a larger repertoire of preexisting temporal firing sequences in the hippocampal cellular...
Insights into bacterial CO2 metabolism revealed by the characterization of four carbonic anhydrases in Ralstonia eutropha H16 - Gai, Claudia S.; Lu, Jingnan; Brigham, Christopher J.; de Carvalho Bernardi, Amanda; Sinskey, Anthony J.
Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium’s capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle. Analysis of the R. eutropha H16 genome sequence revealed the presence of four CA genes: can, can2, caa and cag. We evaluated the importance of each of the CAs in the metabolism of R. eutropha by examination of...
Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome - Flynn, Ryan A.; Almada, Albert Ernesto; Zamudio, Jesse Ray; Sharp, Phillip A.
Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5′ termini and heterogeneous nonpolyadenylated 3′ ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1–4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs...