Difference: DinshawPatel (1 vs. 7)

Abstract: This abstract is not available.

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Thesaurus Terms: RNA, infection base, cell, complementary RNA, gene, human, immune response, insight, phosphate, plant, protein, small interfering RNA, suppression, tissue, transfection, university, virus

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2009 Department: Project Start: 01-MAR-2006 Project End: 28-FEB-2011 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: MSFC

Abstract: DESCRIPTION (provided by applicant): Genomic integrity depends critically on the fidelity and efficiency of DNA replication. Processive polymerases can stall at DNA damage sites and translesion synthesis is then dominated by bypass polymerases, involving error-free (mutation-avoiding) or error-prone (mutation-generating) pathways. Formation of oxidative damage sites are accelarated under conditions of smoking-induced oxidative stress, and markedly increased levels of oxidative damage adducts have been detected in lung tissue that has been chronically exposed to cigarette smoke. Our goal is to understand the molecular interactions that define the mutagenic spectrum associated with replication of oxidative damage sites by bypass polymerases. Our proposed crystallographic studies will be undertaken on the most prevalent oxidative damage lesions, namely, 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct and the fused bicyclic spiroiminodihydantoin adduct, positioned at template-primer junctions, as part of binary and ternary (with incoming nucleoside triphosphates) complexes, with the thermophilic Dpo4 bypass polymerase. We have already solved crystal structures of binary and ternary complexes of oxoG- containing template-primer junction, Dpo4 and incoming dCTP that have provided detailed insights into dCTP-binding and dCTP-incorporation steps, and following additional planned experiments, elongation steps. Our proposed structural studies of Dpo4 ternary complexes of oxidative guanine adducts in the C- *G-C sequence context will be extended to the C-*G-T sequence context, which constitute a sub-set of mutational 'hot spots' in the p53 tumor suppressor gene. Our efforts should elucidate the geometric fit, alignment and register for individual oxidative damage lesions of varying size and shape positioned in the active site of Dpo4, should determine the specific interactions and pairings of the lesion site with complementary and non-cognate incoming nucleoside triphosphates, and should identify key residues and alignments for facilitating the divalent cation-mediated nucleotidyl transfer reaction. The proposed studies should provide structural insights into how bypass is modulated by lesion architecture and base sequence context, and provide explanations for the distribution of point mutations relative to frameshift deletions.

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Thesaurus Terms: DNA damage, DNA directed DNA polymerase, enzyme activity, oxidative stress, protein structure function adduct, guanine, hydantoin, nitric oxide, peroxynitrite, purine, stereoisomer X ray crystallography

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 05-MAR-1988 Project End: 31-JUL-2010 ICD: NATIONAL CANCER INSTITUTE IRG: CE

Abstract: DESCRIPTION (provided by applicant): A number of cancer-related autoimmune and neurologic diseases are associated with RNA-binding proteins. This revised application focuses on a structural (x-ray and NMR) and functional (impact of mutations) investigation of protein-RNA recognition in Fragile X mental retardation (FXMR) and paraneoplastic opsoclonus-myoclonus ataxia (POMA) syndromes. This project represents a collaborative effort with the Robert Darnell laboratory, which has biochemically identified the relevant protein-RNA complexes, and is currently addressing biological and clinical issues associated with these syndromes. Project 1: Our structural efforts on the FXMR syndrome have focused on the complex between an RGG peptide and a quadruplex-duplex neuronal RNA scaffold for which we have obtained exceptional NMR spectra, including spectra of samples uniformly 13C,15N-labeled in the peptide and RNA components. These structural efforts will be followed up by mutational experiments coupled with affinity measurements using surface plasmon resonance to identify energetic contributions involving key residues associated with molecular recognition. A new project involves the structure determination of a complex between one of the two FXMR KH domains and its in vitro-selected RNA target identified in the Darnell laboratory. We also propose to solve structures of three distinct sets of crystals of FXMR syndrome r(CGG)n repeats, n = 3, which have been grown in various space groups, one of which diffracts to 1.0 A resolution. Project 2: Our proposed structural studies of the POMA syndrome are directed towards our long-term goal of providing a structural understanding of how full length Nova (KH1-KH2-KH3) proteins target and regulate alternative splicing events within the alpha-2 glycine receptor subunit pre-mRNA, where the RNA target contains the UCAU-Y-UCAU-Y-UCAU sequence. We have already determined a crystal structure ol a Nova KH1/KH2 construct bound to its UCAN-UCAN-containing RNA hairpin, where KH1 targets one of the two UCAN RNA segments, while KH2 is involved in protein dimerization. We are currently mutating residues at the unanticipated KH dimeric interface to evaluate its functional role, as well as attempting to provide a molecular explanation for protein engineering efforts aimed at extending the KH-RNA interface.

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Thesaurus Terms: DNA damage, RNA, RNA binding protein, ataxia, chemical association, fragile X syndrome, neural degeneration, nucleic acid structure, peptide structure, protein structure function molecular dynamics, neoplastic transformation, structural biology crystallization, nuclear magnetic resonance spectroscopy

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 05-DEC-1989 Project End: 30-JUN-2009 ICD: NATIONAL CANCER INSTITUTE IRG: BBCA

Abstract: DESCRIPTION (provided by applicant): Glycosphingolipids (GSL)-enriched 'rafts' and caveolae are membrane microdomains that putatively function as lateral organizing sites for signaling proteins involved in oncogenesis. Because the overexpression of select caveolar proteins, i.e. caveolin-1, is associated with tumor cell survival, aggression and metastatic potential, targeting GSL-enriched caveolae may prove useful as a new therapy for the functional disruption of metastatis, tumorigenesis, and tumor progression. The processes by which GSL-enriched domains are formed and maintained are not well defined but are expected to involve specific proteins that can bind and transfer GSLs between and within cells. Our objective is to elucidate the structure of human glycolipid transfer protein (GLTP) and related orthologs and to identify/-characterize folding domains responsible for glycolipid liganding selectivity using structural, dynamical, and mutational approaches by taking advantage of the structural expertise of the Dinshaw Patel (Sloan-Kettering Institute, NY) and the molecular biological and glycosphingolipid expertise of the Rhoderick Brown (Hormel Institute, MN) laboratories. The rationale for the research is that, solving the structure of GLTP and related orthologs in their apo and glycolipid-liganded states will enable mapping of the protein domains and associated key amino acid residues involved in GLTP functionality. Acquiring this knowledge will provide a foundation for pursuing the future development of pharmacologic agents that can specifically target GLTP in oncogenic cells displaying aberrant GLTP activity. The proposed work is innovative because it capitalizes on the first-ever, structural insights into human GLTP. The novel two-layer, all alpha helical topology of GLTP differs distinctly from the folding topologies of other known lipid binding/transfer proteins, suggesting that the GLTP folding motif defines a novel family of proteins. The studies will take advantage of our recent successes in the molecular cloning and expression of GLTP and related point mutants. It is our expectation that the proposed structural-dynamics-mutational studies of human GLTPs and related orthologs will provide unparalleled insights into the functional workings of this emerging new protein family. This new knowledge is expected to be significant by providing a foundation for using GLTP in new and innovative ways, such as introducing specific GSL antigens into cancer cells to help achieve targeted destruction of diseased cells via immunotherapeutic means.

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Thesaurus Terms: HIV infection, Herpesviridae, Herpesviridae disease, Kaposi's sarcoma, pathologic process, virus load diagnosis quality /standard, disease /disorder etiology, longitudinal human study, serology /serodiagnosis, virus DNA clinical research, human subject, polymerase chain reaction

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-JUN-2006 Project End: 30-APR-2011 ICD: NATIONAL CANCER INSTITUTE IRG: BBM

Abstract: DESCRIPTION (provided by applicant): This revised application focuses on 3 projects addressing structural and functional aspects of folding, recognition and catalysis by RNA regulatory domains. Project 1 focuses on the structural characterization of the core domains of metabolite-sensing mRNAs discovered in the Ronald Breaker laboratory, which adopt complex junctional topologies capable of ligand-induced functional modulation. Our structural research will initially focus on the free and bound core domains of guanine/adenine-sensing and thiamine pyrophosphate-sensing mRNAs, for which we have collected promising NMR and crystallographic data. Our structural studies combined with functional efforts in the Breaker laboratory should highlight the principles of molecular recognition and metabolite encapsulation by mRNA, and define the allosteric mRNA transitions associated with the modulation of gene expression levels and metabolic homeostasis. Project 2 on ribozymes catalyzing chemical reactions, focuses on the structural characterization of RNA motifs with Diels-Alderase catalytic activities discovered in the Andres Jaschke laboratory. We are attempting to structurally characterize the catalytic RNA scaffold in the context of bound substrates, transition state analogs and products, with promising crystallographic and NMR data collected on the product complex. Our structural characterization of the Diels-Alderase ribozyme, together with mutational and energetics studies in the Jaschke laboratory, should identify principles for generation of novel ribozymes with controllable catalytic activities, tunable specificites and enantiomeric capabilities. Project 3 focuses on protein-RNA recognition events that mediate the degradation of metazoan histone mRNA, a process tightly coupled to cell cycle progression. Histone mRNAs contain a unique bipartite stem-loop scaffold followed by an ACCA sequence, whose stem, loop and flanking sequences are targeted along opposite faces, both separately and simutaenously, by a stem-loop binding protein and a histone mRNA 3' end-specific exonuclease. Our proposed structural and energetics characterization of binary and ternary protein-RNA recognition events involved in histone mRNA 3'-end recognition and cleavage should provide insights into the mechanisms that cells use to achieve precise cell cycle-regulated mRNA degradation.

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Thesaurus Terms: bioenergetics, catalyst, chemical binding, chemical structure function, gene expression, intermolecular interaction, messenger RNA, nucleic acid sequence adenine, allosteric site, cell cycle, chemical cleavage, gene mutation, guanine, histone, protein degradation, ribozyme, thiamine pyrophosphate X ray crystallography, calorimetry, nuclear magnetic resonance spectroscopy, surface plasmon resonance

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-APR-1988 Project End: 31-DEC-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: PB