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1.
Cancer Cell ; 17(4): 400-11, 2010 Apr 13.
Article in English | MEDLINE | ID: mdl-20385364

ABSTRACT

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.


Subject(s)
Lymphoma, Large B-Cell, Diffuse/pathology , Proto-Oncogene Proteins/antagonists & inhibitors , Repressor Proteins/antagonists & inhibitors , Animals , Cell Division , Cell Survival/drug effects , Crystallography, X-Ray , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/toxicity , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/toxicity , Transcription, Genetic , Zinc Fingers
2.
Mol Cell ; 29(3): 384-91, 2008 Feb 15.
Article in English | MEDLINE | ID: mdl-18280243

ABSTRACT

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.


Subject(s)
Peptides/metabolism , Proto-Oncogene Proteins c-bcl-6/chemistry , Proto-Oncogene Proteins c-bcl-6/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Dimerization , Histidine/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Mutation , Peptides/isolation & purification , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-6/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-6/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Sequence Alignment , Thioredoxins/metabolism
3.
EMBO J ; 22(23): 6346-55, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14633993

ABSTRACT

The protein FinO represses F-plasmid conjugative transfer by facilitating interactions between the mRNA of the major F-plasmid transcriptional activator, TraJ, and an antisense RNA, FinP. FinO is known to bind stem-loop structures in both FinP and traJ RNAs; however, the mechanism by which FinO facilitates sense-antisense pairing is poorly understood. Here we show that FinO acts as an RNA chaperone to promote strand exchange and duplexing between minimal RNA targets derived from FinP. This strongly suggests that FinO may function to destabilize internal secondary structures within FinP and traJ RNAs that would otherwise act as a kinetic trap to sense-antisense pairing. The energy for FinO-catalyzed base-pair destabilization does not arise from ATP hydrolysis but appears to be supplied directly from FinO RNA binding free energy. An analysis of the activities of mutants that are specifically deficient in strand exchange but not RNA-binding activity demonstrates that strand exchange is essential to the ability of FinO to mediate sense-antisense RNA recognition, and that this function also plays a role in repression of conjugation in vivo.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , RNA, Antisense/metabolism , RNA, Bacterial/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Base Sequence , Catalysis , Escherichia coli/genetics , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes , RNA, Antisense/chemistry , RNA, Bacterial/chemistry
4.
J Biol Chem ; 278(30): 27663-71, 2003 Jul 25.
Article in English | MEDLINE | ID: mdl-12748195

ABSTRACT

F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.


Subject(s)
Conjugation, Genetic/genetics , Escherichia coli Proteins/genetics , F Factor/genetics , RNA-Binding Proteins/genetics , RNA/metabolism , Repressor Proteins/genetics , Base Sequence , Blotting, Northern , Immunoblotting , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids/metabolism , RNA/chemistry , RNA, Antisense/metabolism , RNA, Messenger/metabolism , Transcription, Genetic
5.
RNA ; 8(6): 816-23, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12088153

ABSTRACT

The conjugative transfer of F-plasmids is repressed by a two-component system, which consists of the antisense RNA FinP and the protein FinO. FinO binds FinP, protecting it from endonucleolytic degradation and facilitating duplex formation between FinP and its complementary RNA. Here we present the results of site-specific protein-RNA cross-linking and gel-based fluorescence resonance energy transfer (gelFRET) experiments used to probe the structure of a complex of FinO bound to an RNA target consisting of a duplex with 5' and 3' single-stranded tails. The crosslinking experiments reveal that an extensive, largely positively charged surface on FinO contacts RNA. The gelFRET measurements indicate that the 5' single-stranded tail of the RNA is in closer contact with much of the protein than the distal, blunt end of the RNA duplex. These data suggest that significant conformational adjustments in the protein and/or the RNA accompany complex formation.


Subject(s)
Bacterial Proteins/genetics , Cross-Linking Reagents/chemistry , Escherichia coli Proteins , RNA-Binding Proteins/genetics , RNA/chemistry , Repressor Proteins , Bacterial Proteins/chemistry , Base Sequence , DNA Primers , F Factor , Molecular Probes , Point Mutation , RNA-Binding Proteins/chemistry
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