30 results on '"Assa-Munt N"'
Search Results
2. DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.
- Author
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Gaal, T, primary, Ross, W, additional, Blatter, E E, additional, Tang, H, additional, Jia, X, additional, Krishnan, V V, additional, Assa-Munt, N, additional, Ebright, R H, additional, and Gourse, R L, additional
- Published
- 1996
- Full Text
- View/download PDF
3. The solution structure of the Oct-1 POU-specific domain reveals a striking similarity to the bacteriophage λ repressor DNA-binding domain
- Author
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Assa-Munt, N, primary
- Published
- 1993
- Full Text
- View/download PDF
4. Mapping the anatomy of the immunodominant domain of the human immunodeficiency virus gp41 transmembrane protein: peptide conformation analysis using monoclonal antibodies and proton nuclear magnetic resonance spectroscopy
- Author
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Oldstone, M B, primary, Tishon, A, additional, Lewicki, H, additional, Dyson, H J, additional, Feher, V A, additional, Assa-Munt, N, additional, and Wright, P E, additional
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- 1991
- Full Text
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5. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation.
- Author
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McInerney, E M, Rose, D W, Flynn, S E, Westin, S, Mullen, T M, Krones, A, Inostroza, J, Torchia, J, Nolte, R T, Assa-Munt, N, Milburn, M V, Glass, C K, and Rosenfeld, M G
- Abstract
Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
- Published
- 1998
6. A single BIR domain of XIAP sufficient for inhibiting caspases.
- Author
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Takahashi, R, Deveraux, Q, Tamm, I, Welsh, K, Assa-Munt, N, Salvesen, G S, and Reed, J C
- Abstract
The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (Ki) for BIR2-mediated inhibition of caspases-3 and -7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.
- Published
- 1998
7. Analysis of apoptosis protein expression in early-stage colorectal cancer suggests opportunities for new prognostic biomarkers.
- Author
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Krajewska M, Kim H, Kim C, Kang H, Welsh K, Matsuzawa S, Tsukamoto M, Thomas RG, Assa-Munt N, Piao Z, Suzuki K, Perucho M, Krajewski S, and Reed JC
- Subjects
- Age Factors, Apoptotic Protease-Activating Factor 1, Biomarkers metabolism, Chemotherapy, Adjuvant, Colonic Neoplasms pathology, Colorectal Neoplasms diagnosis, Female, Humans, Immunoblotting, Immunohistochemistry, Intracellular Signaling Peptides and Proteins metabolism, Male, Microsatellite Repeats, Multivariate Analysis, Oligonucleotide Array Sequence Analysis, Phenotype, Prognosis, Proportional Hazards Models, Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Retrospective Studies, Sex Factors, Time Factors, Tissue Distribution, Treatment Outcome, Apoptosis, Biomarkers, Tumor metabolism, Colorectal Neoplasms metabolism, Colorectal Neoplasms pathology
- Abstract
Purpose: Although most stage II colon cancers are potentially curable by surgery alone, approximately 20% of patients relapse, suggesting a need for establishing prognostic markers that can identify patients who may benefit from adjuvant chemotherapy. We tested the hypothesis that differences in expression of apoptosis-regulating proteins account for differences in clinical outcome among patients with early-stage colorectal cancer., Experimental Design: Tissue microarray technology was employed to assay the expression of apoptosis-regulating proteins by immunohistochemistry in 106 archival stage II colorectal cancers, making correlations with disease-specific survival. The influence of microsatellite instability (MSI), tumor location (left versus right side), patient age, and gender was also examined., Results: Elevated expression of several apoptosis regulators significantly correlated with either shorter (cIAP2; TUCAN) or longer (Apaf1; Bcl-2) overall survival in univariate and multivariate analyses. These biomarkers retained prognostic significance when adjusting for MSI, tumor location, patient age, and gender. Moreover, certain combinations of apoptosis biomarkers were highly predictive of death risk from cancer. For example, 97% of patients with favorable tumor phenotype of cIAP2(low) plus TUCAN(low) were alive at 5 years compared with 60% of other patients (P = 0.00003). In contrast, only 37% of patients with adverse biomarkers (Apaf1(low) plus TUCAN(high)) survived compared with 83% of others at 5 years after diagnosis (P< 0.0001)., Conclusions: Immunohistochemical assays directed at detection of certain combinations of apoptosis proteins may provide prognostic information for patients with early-stage colorectal cancer, and therefore could help to identify patients who might benefit from adjuvant chemotherapy or who should be spared it.
- Published
- 2005
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8. 1H, 15N and 13C resonance assignments of the C345C domain of the complement component C5.
- Author
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Bramham J, Rance M, Thai CT, Uhrín D, Assa-Munt N, Ogata RT, and Barlow PN
- Subjects
- Escherichia coli chemistry, Humans, Recombinant Proteins chemistry, Carbon Isotopes chemistry, Complement C5 chemistry, Magnetic Resonance Spectroscopy, Nitrogen Isotopes chemistry, Software
- Published
- 2004
- Full Text
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9. Identification of a PU.1-IRF4 protein interaction surface predicted by chemical exchange line broadening.
- Author
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McKercher SR, Lombardo CR, Bobkov A, Jia X, and Assa-Munt N
- Subjects
- Binding Sites, DNA metabolism, Fluorescence, Interferon Regulatory Factors, Mutation, Protein Conformation, DNA-Binding Proteins chemistry, Proto-Oncogene Proteins chemistry, Trans-Activators chemistry, Transcription Factors chemistry
- Abstract
Relaxation values reflecting residue-specific line broadening revealed amino acids in the DNA-binding domain of PU.1 on a surface potentially involved in protein-protein interactions. Mutation of these amino acids did not cause protein unfolding but destabilized PU.1-DNA binding. Addition of IFN response factor 4 to form the ternary complex recovered binding stability. Fluorescence quenching experiments proved that this surface of PU.1 interacts with IFN response factor 4 during binding. Our results provide evidence that residues that display increased conformational exchange can be used to predict areas of protein-protein interactions.
- Published
- 2003
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10. NMR assignment of human ASC2, a self contained protein interaction domain involved in apoptosis and inflammation.
- Author
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Espejo F, Green M, Preece NE, and Assa-Munt N
- Subjects
- Humans, Nuclear Receptor Coactivators, Protein Binding, Protein Structure, Tertiary, Structure-Activity Relationship, Apoptosis, Inflammation, Intracellular Signaling Peptides and Proteins, Nuclear Magnetic Resonance, Biomolecular, Transcription Factors chemistry, Transcription Factors metabolism
- Published
- 2002
- Full Text
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11. Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3.
- Author
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Smith JW, Le Calvez H, Parra-Gessert L, Preece NE, Jia X, and Assa-Munt N
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Bacteriophages metabolism, Binding Sites, Binding, Competitive, Calcium metabolism, Dose-Response Relationship, Drug, Enzyme-Linked Immunosorbent Assay, Gene Library, Humans, Inhibitory Concentration 50, Magnesium metabolism, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Oligopeptides metabolism, Peptides chemistry, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Blood Platelets metabolism, Ions, Ligands, Platelet Glycoprotein GPIIb-IIIa Complex chemistry, Platelet Glycoprotein GPIIb-IIIa Complex metabolism
- Abstract
Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca(2+) and Mg(2+) bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin alpha(IIb)beta(3). The library was used to select for peptides that bind to the integrin alpha(IIb)beta(3) preferentially in Ca(2+) versus Mg(2+). Peptides were identified which bound selectively in each ion. The Ca(2+)-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg(2+)-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg(2+)-selected peptides contained a unique basic patch that protrudes from the base of the turn.
- Published
- 2002
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12. Some insights into protein structural class prediction.
- Author
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Zhou GP and Assa-Munt N
- Subjects
- Amino Acids chemistry, Bayes Theorem, Forecasting, Algorithms, Models, Statistical, Proteins chemistry
- Abstract
It has been quite clear that the success rate for predicting protein structural class can be improved significantly by using the algorithms that incorporate the coupling effect among different amino acid components of a protein. However, there is still a lot of confusion in understanding the relationship of these advanced algorithms, such as the least Mahalanobis distance algorithm, the component-coupled algorithm, and the Bayes decision rule. In this communication, a simple, rigorous derivation is provided to prove that the Bayes decision rule introduced recently for protein structural class prediction is completely the same as the earlier component-coupled algorithm. Meanwhile, it is also very clear from the derivative equations that the least Mahalanobis distance algorithm is an approximation of the component-coupled algorithm, also named as the covariant-discriminant algorithm introduced by Chou and Elrod in protein subcellular location prediction (Protein Engineering, 1999; 12:107-118). Clarification of the confusion will help use these powerful algorithms effectively and correctly interpret the results obtained by them, so as to conduce to the further development not only in the structural prediction area, but in some other relevant areas in protein science as well., (Copyright 2001 Wiley-Liss, Inc.)
- Published
- 2001
- Full Text
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13. Solution structures and integrin binding activities of an RGD peptide with two isomers.
- Author
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Assa-Munt N, Jia X, Laakkonen P, and Ruoslahti E
- Subjects
- Bacteriophages metabolism, Disulfides chemistry, Humans, Integrin alpha4beta1, Jurkat Cells, Nuclear Magnetic Resonance, Biomolecular, Peptide Library, Protein Binding, Protein Conformation, Protein Isoforms chemistry, Protein Isoforms metabolism, Receptors, Lymphocyte Homing metabolism, Receptors, Vitronectin metabolism, Solutions, Tumor Cells, Cultured, Integrins metabolism, Oligopeptides chemistry, Oligopeptides metabolism
- Abstract
The Arg-Gly-Asp (RGD) sequence serves as the primary integrin recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the activities of the matrix proteins. Depending on the context of the RGD sequence, an RGD-containing peptide may bind to all of the RGD-directed integrins, to a few, or to only a single one. We have previously isolated from a phage-displayed peptide library a cyclic peptide that binds avidly to the alpha(v)beta3 and alpha(v)beta5 integrins but does not bind to other closely related integrins. This peptide, ACDCRGDCFCG, exists in two natural configurations depending on internal disulfide bonding. The peptide with the 1-4; 2-3 disulfide bond arrangement accounts for most of the alpha(v) integrin binding activity, whereas the 1-3; 2-4 peptide is about 10-fold less potent. Solution structure analysis by nuclear magnetic resonance reveals an entirely different presentation of the RGD motif in the two isomers of RGD-4C. These results provide new insight into the ligand recognition specificity of integrins.
- Published
- 2001
- Full Text
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14. Neurotrophin dependence domain: a domain required for the mediation of apoptosis by the p75 neurotrophin receptor.
- Author
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Rabizadeh S, Ye X, Sperandio S, Wang JJ, Ellerby HM, Ellerby LM, Giza C, Andrusiak RL, Frankowski H, Yaron Y, Moayeri NN, Rovelli G, Evans CJ, Butcher LL, Nolan GP, Assa-Munt N, and Bredesen DE
- Subjects
- Amino Acid Sequence genetics, Animals, Cell-Free System metabolism, Dimerization, Genetic Vectors genetics, Humans, Mutation genetics, Peptide Fragments genetics, Plasmids biosynthesis, Plasmids genetics, Protein Structure, Tertiary genetics, Receptor, Nerve Growth Factor genetics, Recombinant Fusion Proteins genetics, Transfection, Tumor Cells, Cultured cytology, Tumor Cells, Cultured metabolism, Apoptosis genetics, Receptor, Nerve Growth Factor chemistry, Receptor, Nerve Growth Factor metabolism
- Abstract
The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.
- Published
- 2000
- Full Text
- View/download PDF
15. Dimerization-dependent block of the proapoptotic effect of p75(NTR).
- Author
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Wang JJ, Rabizadeh S, Tasinato A, Sperandio S, Ye X, Green M, Assa-Munt N, Spencer D, and Bredesen DE
- Subjects
- Apoptosis drug effects, Carrier Proteins drug effects, Cells, Cultured, Cross-Linking Reagents pharmacology, Dimerization, Humans, Nerve Tissue Proteins drug effects, Neurotrophin 3 pharmacology, Protein Structure, Tertiary drug effects, Protein Structure, Tertiary physiology, Receptors, Nerve Growth Factor drug effects, Selective Estrogen Receptor Modulators pharmacology, Tacrolimus analogs & derivatives, Tacrolimus pharmacology, Tamoxifen pharmacology, Transfection, Apoptosis physiology, Carrier Proteins metabolism, Nerve Tissue Proteins metabolism, Receptors, Growth Factor, Receptors, Nerve Growth Factor metabolism
- Abstract
The biochemical mechanism by which neurons become dependent on neurotrophins for survival is unknown. We found previously that the common neurotrophin receptor, p75(NTR), is a mediator of neurotrophin dependence and that this effect requires a novel type of domain dubbed a neurotrophin dependence domain. We report here that, in contrast to other proapoptotic receptors such as Fas, apoptosis induction by p75(NTR) requires monomerization, with dimerization inhibiting the effect. Blocking the proapoptotic effect of the monomer by dimerization requires a distinct domain that lies at the carboxyterminus of p75(NTR). These results define a novel type of domain required for inhibiting apoptosis induction by p75(NTR)., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
- Full Text
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16. Backbone dynamics of a short PU.1 ETS domain.
- Author
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Jia X, Lee LK, Light J, Palmer AG 3rd, and Assa-Munt N
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Conserved Sequence, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Deuterium metabolism, Diffusion, Hydrogen metabolism, Kinetics, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Folding, Protein Structure, Secondary, Protons, Solvents, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments chemistry, Peptide Fragments metabolism, Proto-Oncogene Proteins chemistry, Proto-Oncogene Proteins metabolism, Trans-Activators chemistry, Trans-Activators metabolism
- Abstract
The resonance assignments, secondary structure and backbone dynamics of the ETS domain of the transcription factor PU.1 have been determined for the free protein in solution by NMR spectroscopy. The secondary structure for the free ETS domain is similar to that observed in the crystal structure of the PU.1 protein complexed with DNA, except that helix alpha2 and recognition helix alpha3 are shorter for the free protein in solution. Backbone dynamics of the protein have been examined using amide hydrogen-deuterium exchange and (15)N laboratory-frame spin relaxation measurements. A significant probability of local unfolding of helix alpha2, which precedes the loop-helix-loop DNA recognition domain, is inferred from the very fast hydrogen-deuterium exchange for amide protons in this helix. The (15)N relaxation measurements indicate that the protein is partially oligomerized at a concentration of 2.5 mM, but monomeric at a concentration of 0.3 mM. The (15)N relaxation data for the low concentration sample were interpreted, using the model-free formalism, to provide insight into protein dynamics on picosecond-nanosecond and microsecond-millisecond time scales. High flexibility of the protein backbone is observed for the residues in the loop between alpha2 and alpha3. This loop is variable in length and in structure within the class of winged helix proteins and is partially responsible for binding to DNA. The dynamic properties observed for alpha2, alpha3 and the intervening loop may indicate a correlation between protein plasticity in particular structural elements and recognition of specific DNA sequences., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
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17. Mutation analysis of the Pip interaction domain reveals critical residues for protein-protein interactions.
- Author
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Ortiz MA, Light J, Maki RA, and Assa-Munt N
- Subjects
- Amino Acid Sequence, Animals, Binding Sites genetics, DNA Mutational Analysis, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Interferon Regulatory Factors, Molecular Sequence Data, Protein Binding, Proto-Oncogene Proteins metabolism, Trans-Activators genetics, Trans-Activators metabolism, DNA-Binding Proteins chemistry, Proto-Oncogene Proteins chemistry, Trans-Activators chemistry
- Abstract
The PU.1 interaction partner (Pip) is a member of the interferon regulatory factor family that regulates gene expression through heterodimerization with the ETS transcription factor PU.1. Binding of Pip alone to DNA is weak, and usually it is recruited by phosphorylated PU.1 to form a strong ternary complex with specific DNA sequences. An approach combining sequence homology analysis, secondary structure predictions, and a precise mutational strategy has been used to determine critical residues within the Pip heterodimerization domain that contribute to ternary complex formation. We have delimited the Pip interaction domain to residues 245-422 by using deletion analysis. Site-directed mutagenesis of conserved polar amino acids within two predicted alpha-helices contained in this region, and which are highly conserved in the IRF family, confirmed the importance of these residues for Pip-PU.1 interaction with DNA as well as for trans-activation activity. Our results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.
- Published
- 1999
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18. The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis.
- Author
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Mehlen P, Rabizadeh S, Snipas SJ, Assa-Munt N, Salvesen GS, and Bredesen DE
- Subjects
- Animals, Aspartic Acid metabolism, Caco-2 Cells, Caspase 7, Caspases metabolism, Cell Adhesion Molecules physiology, Cell Line, DCC Receptor, Humans, Mice, Mutagenesis, Site-Directed, Nerve Growth Factors metabolism, Netrin-1, Receptors, Cell Surface genetics, Receptors, Cell Surface physiology, Transfection, Apoptosis genetics, Cell Adhesion Molecules genetics, Genes, DCC, Genes, Tumor Suppressor, Tumor Suppressor Proteins
- Abstract
The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.
- Published
- 1998
- Full Text
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19. p75NTR and the concept of cellular dependence: seeing how the other half die.
- Author
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Bredesen DE, Ye X, Tasinato A, Sperandio S, Wang JJ, Assa-Munt N, and Rabizadeh S
- Subjects
- Animals, Cell Line, Cytoplasm metabolism, Ligands, Nerve Growth Factors deficiency, Neurons metabolism, Receptor Protein-Tyrosine Kinases metabolism, Receptor, Ciliary Neurotrophic Factor, Receptor, Nerve Growth Factor, Receptors, Nerve Growth Factor biosynthesis, Receptors, Nerve Growth Factor genetics, Receptors, Nerve Growth Factor metabolism, Apoptosis physiology, Nerve Growth Factors metabolism, Receptors, Nerve Growth Factor physiology, Signal Transduction physiology
- Abstract
Cells depend on specific stimuli, such as trophic factors, for survival and in the absence of such stimuli, undergo apoptosis. How do cells initiate apoptosis in response to the withdrawal of trophic factors or other dependent stimuli? Recent studies of apoptosis induction by neurotrophin withdrawal argue for a novel form of pro-apoptotic signal transduction - 'negative signal transduction' - in which the absence of ligand-receptor interaction induces cell death. We have found that the prototype for this form of signaling - the common neurotrophin receptor, p75NTR - creates a state of cellular dependence (or addiction) on neurotrophins, and that this effect requires an 'addiction/dependence domain' (ADD) in the intracytoplasmic region of p75NTR. We have recently found other receptors that include dependence domains, arguing that dependence receptors, and their associated dependence domains, may be involved in a rather general mechanism to create cellular states of dependence on trophic factors, cytokines, adhesion, electrical activity and other dependent stimuli.
- Published
- 1998
- Full Text
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20. Solution structure of Compstatin, a potent complement inhibitor.
- Author
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Morikis D, Assa-Munt N, Sahu A, and Lambris JD
- Subjects
- Binding, Competitive, Disulfides, Humans, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Conformation, Solutions, Structure-Activity Relationship, Complement C3 chemistry, Complement Inactivator Proteins metabolism, Complement Inactivator Proteins ultrastructure, Peptides, Cyclic metabolism
- Abstract
The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13-residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure-function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry-restrained simulated-annealing methodology, using distance, dihedral angle, and 3J(NH-Halpha)-coupling constant restraints. The Compstatin structure contained a type I beta-turn comprising the segment Gln5-Asp6-Trp7-Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3J(NH-Halpha)-coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100-fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side-chain interactions and the beta-turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.
- Published
- 1998
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21. A cytoplasmic peptide of the neurotrophin receptor p75NTR: induction of apoptosis and NMR determined helical conformation.
- Author
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Hileman MR, Chapman BS, Rabizadeh S, Krishnan VV, Bredesen D, Assa-Munt N, and Plesniak LA
- Subjects
- Cyclic N-Oxides pharmacology, Humans, Lipid Metabolism, Magnetic Resonance Spectroscopy, Micelles, Peptide Fragments chemical synthesis, Peptide Fragments pharmacology, Protein Conformation, Receptor, Nerve Growth Factor, Receptors, Nerve Growth Factor metabolism, Spin Labels, Structure-Activity Relationship, Temperature, Tumor Cells, Cultured, Apoptosis, Peptide Fragments chemistry, Protein Structure, Secondary, Receptors, Nerve Growth Factor chemistry
- Abstract
The neurotrophin receptor (NTR) and tumor necrosis factor receptor family of receptors regulate apoptotic cell death during development and in adult tissues [Beutler and van Huffel, Science 264 (1994) 667-668]. We have examined a fragment of p75NTR from the carboxyl terminus of the receptor and a variant form of this peptide via NMR techniques and in vitro assays for apoptotic activity. The wild type peptide induces apoptosis and adopts a helical conformation oriented parallel to the surface of lipid micelles, whereas the variant form adopts a non-helical conformation in the presence of lipid and shows no activity. These experiments suggest a link between structure and function of the two peptides.
- Published
- 1997
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22. Expression cloning of cDNA by phage display selection.
- Author
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Light J, Maki R, and Assa-Munt N
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, DNA, Complementary, Immunoglobulin Fab Fragments immunology, Immunoglobulin kappa-Chains biosynthesis, Immunoglobulin kappa-Chains immunology, Leucine Zippers genetics, Mice, Molecular Sequence Data, Bacteriophages genetics, Cloning, Molecular methods, Immunoglobulin kappa-Chains genetics, Peptide Library
- Abstract
Expression cloning of a mouse kappa chain fragment has been achieved from a cDNA library by display of expressed proteins on filamentous phage and affinity selection for binding to anti-mouse Fab antibodies. Expressed proteins were anchored to the phage coat by a synthetic, anti-parallel leucine zipper, which had been selected from a semi-randomized zipper library for the ability to connect a test protein to phage. From a library of 4 x 10(6) transformants, two separate clones displaying different size cDNA inserts were recovered after four selection rounds. These results further demonstrate the utility of phage display for cDNA expression cloning.
- Published
- 1996
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23. Solution conformation of an immunogenic peptide derived from the principal neutralizing determinant of the HIV-2 envelope glycoprotein gp125.
- Author
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Campbell AP, Sykes BD, Norrby E, Assa-Munt N, and Dyson HJ
- Subjects
- AIDS Vaccines chemistry, Amino Acid Sequence, Disulfides chemistry, Drug Design, Gene Products, env genetics, HIV Antigens genetics, HIV-2 genetics, Humans, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Molecular Structure, Neutralization Tests, Peptides genetics, Protein Conformation, Protein Precursors genetics, Protein Structure, Secondary, Stereoisomerism, Vaccines, Synthetic chemistry, env Gene Products, Human Immunodeficiency Virus, Gene Products, env chemistry, Gene Products, env immunology, HIV Antigens chemistry, HIV-2 chemistry, HIV-2 immunology, Peptides chemistry, Peptides immunology, Protein Precursors chemistry, Protein Precursors immunology
- Abstract
Background: The conformational preferences of a number of peptides with sequences related to the envelope glycoproteins of HIV-1 have been investigated in the past few years. Similar studies have not been made for HIV-2, which is a distinct virus with similar physiological effects to those of HIV-1. The discovery of common structural features would be a promising route to the design of immunogens for generally effective HIV vaccines. We present the results of an NMR conformational study of a sequence deriving from the V3 loop of HIV-2., Results: Three synthetic immunogenic peptides were studied, of 12, 22 and 39 amino acids in length, all containing a central Met-Ser-Gly-Arg sequence conserved among a number of HIV-2 isolates. In addition, the 39-mer contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to comprise an important structural and immunological component of the intact glycoprotein. All three peptides display well defined beta-turns in the Met-Ser-Gly-Arg sequence, independent of the integrity of the disulfide bond. No other conformational preferences for folded conformations were found for the peptides., Conclusions: The presence of a beta-turn in the Met-Ser-Gly-Arg sequence is strikingly similar to the behavior seen for the corresponding principal neutralizing determinant sequence from gp120 of HIV-1 and argues, in the absence of information of the three-dimensional structure of the intact proteins, for a similarity in the structure of this region that could be exploited in the design of synthetic peptide vaccines generally effective against HIV infections.
- Published
- 1996
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24. Nuclear magnetic resonance 15N and 1H resonance assignments and global fold of rusticyanin. Insights into the ligation and acid stability of the blue copper site.
- Author
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Hunt AH, Toy-Palmer A, Assa-Munt N, Cavanagh J, Blake RC 2nd, and Dyson HJ
- Subjects
- Amino Acid Sequence, Azurin chemistry, Bacterial Proteins chemistry, Binding Sites, Copper metabolism, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Structure, Secondary, Azurin analogs & derivatives
- Abstract
Nuclear magnetic resonance assignments are reported at pH approximately 3 for a type 1 ("blue") copper protein, rusticyanin, obtained from the acidophilic organism Thiobacillus ferrooxidans. A combination of homonuclear proton and heteronuclear 15N-edited NMR spectra has been used to assign most of the 1H and 15N resonances of reduced rusticyanin. The copper-binding site is shown by analogy with other blue copper proteins to contain the side-chains of Cys138, His143 and Met148 at the C-terminal end of the sequence and a fourth ligand that is most likely a histidine, His85, consistent with the constitution of other type 1 copper sites. The global fold of the molecule is a compact beta-barrel or beta-sandwich, which contains a high proportion of beta-sheet secondary structure and a hydrophobic core particularly rich in aromatic residues. The copper-binding active site is surrounded by aromatic residues, and many of the resonances of the residues flanking the active site are shifted to unusual values, consistent with the effects of ring currents. The protected nature of the copper site is demonstrated by the large number of amide protons that are persistent in this region in 99% 2H2O solution at pH 3.4. We suggest that the unusual acid stability, both of the protein itself and of the blue copper active site, is a direct result of the protected and highly hydrophobic nature of the active site sequence and contacting loops and the high proportion of secondary structure in the protein.
- Published
- 1994
- Full Text
- View/download PDF
25. The solution structure of the Oct-1 POU-specific domain reveals a striking similarity to the bacteriophage lambda repressor DNA-binding domain.
- Author
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Assa-Munt N, Mortishire-Smith RJ, Aurora R, Herr W, and Wright PE
- Subjects
- Amino Acid Sequence, Bacteriophage lambda, Base Sequence, Computer Graphics, Host Cell Factor C1, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Octamer Transcription Factor-1, POU Domain Factors, Protein Structure, Tertiary, Repressor Proteins chemistry, Sequence Homology, Amino Acid, Solutions, Structure-Activity Relationship, Viral Proteins, Viral Regulatory and Accessory Proteins, DNA-Binding Proteins chemistry, Transcription Factors chemistry
- Abstract
The POU-specific (POUs) domain, in association with a POU-type homeodomain, forms the bipartite DNA-binding POU domain. The solution structure of the Oct-1 POUs domain has been determined by multidimensional nuclear magnetic resonance spectroscopy and consists of four alpha helices surrounding a conserved hydrophobic core. The POUs domain is structurally similar to the DNA-binding domains of the bacteriophage lambda and 434 repressors and 434 Cro. These domains exhibit superimposable helix-turn-helix (HTH) motifs, except that in the POUs domain, the first helix and the linker to the second helix of the motif are extended. The conserved structural features have been used to propose a plausible model for DNA binding by the POUs domain. A human dwarfism mutation that affects positive control in the related POU domain protein Pit-1 maps to the same region of the HTH motif as do positive control mutations in lambda repressor.
- Published
- 1993
- Full Text
- View/download PDF
26. Poly(dA-dT) has a right-handed B conformation in solution: a two-dimensional NMR study.
- Author
-
Assa-Munt N and Kearns DR
- Subjects
- Base Sequence, DNA, Magnetic Resonance Spectroscopy, Nucleic Acid Conformation, Structure-Activity Relationship, Poly dA-dT, Polydeoxyribonucleotides
- Abstract
The structure of poly(dA-dT) molecules in solution has been probed by using two-dimensional nuclear Overhauser effect spectroscopy. Cross-relaxation patterns arising from internucleotide and intranucleotide interactions are used to assign the sugar proton resonances and to deduce various structural features. The numerous proton-proton interactions that are observed indicate that poly(dA-dT) is a right-handed helix with a B-type conformation. Both the adenine and thymine nucleotides are in an anti conformation. Although slight differences in the purine-sugar and pyrimidine-sugar intranucleotide interactions are observed, the large differences in the sugar pucker of the adenine vs. thymine nucleotide suggested by some models [Klug, A., Jack, A., Viswamitra, M. A., Kennard, O., Shakked, Z., & Steitz, T. A. (1979) J. Mol. Biol. 131, 669-680] are not evident in the solution structure of poly(dA-dT). In the low-temperature spectra there is unexpected evidence for cross-strand AH2-AH2 interactions.
- Published
- 1984
- Full Text
- View/download PDF
27. 1H NMR study of the binding of bis(acridines) to d(AT)5.d(AT)5. 2. Dynamic aspects.
- Author
-
Assa-Munt N, Leupin W, Denny WA, and Kearns DR
- Subjects
- Adenine Nucleotides chemistry, Aminacrine chemistry, Hydrogen Bonding, Kinetics, Magnetic Resonance Spectroscopy, Temperature, Thymine Nucleotides chemistry, Acridines chemistry, Intercalating Agents chemistry, Oligodeoxyribonucleotides chemistry
- Abstract
Measurements of the 1H NMR spectra and relaxation rates were used to study the dynamic properties of 9-aminoacridine (9AA) and four bis(acridine) complexes with d(AT)5.d(AT)5. The behavior of the 9AA (monointercalator) and that of C8 (bisintercalator containing an eight-carbon atom linker chain) are entirely similar. For both compounds, the lifetime of the drug in a particular binding site is 2-3 ms at approximately 20 degrees C, and neither affects the A.T base pair opening rates. The complex with C10 (bisintercalator containing a 10-carbon atom linker chain) is slightly more stable than the C8 complex since its estimated binding site lifetime is 5-10 ms at 29 degrees C. Base pairs adjacent to the bound C10 are destabilized, relative to free d(AT)5.d(AT)5, but other base pairs in the C10 complex are little affected. Bis(acridine) pyrazole (BAPY) and bis(acridine) spermine (BAS) considerably stabilize those base pairs that are sandwiched between the two acridine chromophores, but in the BAS complex proton exchange from the two flanking base pairs appears to be accelerated, relative to free d(AT)5.d(AT)5. The lifetime of these drugs in specific binding sites is too long (>10 ms) to be manifested in increased line widths, at least up to 41 degrees C. An important conclusion from this study is that certain bisintercalators rapidly migrate along DNA, despite having large binding constants (K>10(6) M-1). For C8 and C10 complexes, migration rates are little different from those deduced for 9AA. The rigid linker chain in BAPY and the charge interactions in BAS retard migration of these two bisintercalators. These results provide new parameters that are useful in understanding the biochemical and biological properties of these and other bisintercalating drugs.
- Published
- 1985
- Full Text
- View/download PDF
28. 1H NMR study of the binding of Bis(acridines) to d(AT)5.d(AT)5. 1. Mode of binding.
- Author
-
Assa-Munt N, Denny WA, Leupin W, and Kearns DR
- Subjects
- Adenine Nucleotides chemistry, Aminacrine chemistry, Magnetic Resonance Spectroscopy, Thymine Nucleotides chemistry, Acridines chemistry, Intercalating Agents chemistry, Oligodeoxyribonucleotides chemistry
- Abstract
1H NMR has been used to investigate the mode of binding to d(AT)5.d(AT)5 of a series of bis(acridine) derivatives connected by different types of linker chains. The length and character (ionic, aliphatic, rigid, and flexible) of the linker chains are found to have a profound effect on the binding of these derivatives to the DNA. Bis(acridine) derivatives with linker chains shorter than 9 A monointercalate under the conditions used in the NMR study, whereas those bis(acridines) with chains of 9.8 A or longer bisintercalate. We find no evidence for the violation of the so-called neighbor exclusion principle. Although all of the bis(acridines) contain the same chromophores, their NMR spectra clearly demonstrate that they form complexes with d(AT)5.d(AT)5 which have different structures. This emphasizes the important effect that the linker chain has on the structure of the intercalation complex.
- Published
- 1985
- Full Text
- View/download PDF
29. 1H NMR relaxation studies of the hydrogen-bonded imino protons of poly(dA-dT).
- Author
-
Assa-Munt N, Granot J, Behling RW, and Kearns DR
- Subjects
- Hydrogen Bonding, Magnetic Resonance Spectroscopy, Mathematics, Thermodynamics, Thymine, Poly dA-dT, Polydeoxyribonucleotides
- Abstract
Measurements on the thymine imino proton relaxation rates have been used to study various structural and dynamic properties of 53 +/- 15 base pair long poly(dA-dT). Below 10 degrees C, the relaxation is dominated by dipolar magnetic interactions. At 1 degrees C the relaxation of the transverse magnetization is exponential (R2 = 124 s-1), but the relaxation of longitudinal magnetization is highly nonexponential due to spin-diffusion effects (initial decay rate constant of 28 s-1 and a slower rate of approximately 2.5 s-1 after equilibration of spin polarization). Neither a rigid-rod model nor simple wormlike motions can account for the observed low-temperature relaxation behavior. However, when localized internal motions of the base pairs (three-state jump model) are allowed for, a good fit of the experimental data is obtained by using a correlation time for internal motion of 7 X 10(-10) s and an angular displacement of the bases of +/- 32 degrees relative to the helix axis. The observed R2/R1 ratio for the thymine imino proton yields a value of 1.14 +/- 0.08 A for the imino proton nitrogen distance. Nuclear Overhauser effect (NOE) measurements establish that the base pairing in poly(dA-dT) is Watson-Crick in solution and not Hoogsteen. Exchange of the T-imino protons with H2O dominates the longitudinal relaxation above 28 degrees C (activation energy of 17 +/- 2 kcal and an exchange rate of 5 +/- 2 s-1 at 300 K). Similar values have been reported for the A X T base pairs in DNA restriction fragments and for A X U base pairs in poly(A) X poly(U). These observations can be explained by a model in which exchange of T-imino protons occurs as a result of a single base pair opening, with a rate that is approximately independent of nearest-neighbor sequences and DNA length. Our observations appear to be inconsistent with a soliton model of proton exchange.
- Published
- 1984
- Full Text
- View/download PDF
30. Interactions of DNA with divalent metal ions. IV. Competitive studies of Mn2+ binding to AT- and GC-rich DNAs.
- Author
-
Granot J, Assa-Munt N, and Kearns DR
- Subjects
- Clostridium perfringens, Hydrogen Bonding, Magnetic Resonance Spectroscopy, Micrococcus, DNA, Bacterial, Manganese
- Published
- 1982
- Full Text
- View/download PDF
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