Glioblastoma remains one of the most challenging forms of cancer to treat. Here, we develop a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We find mutations with loss of heterozygosity of LZTR-1, an adaptor of Cul3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR-1 function, which restrains self-renewal and growth of glioma spheres retaining stem cell features. Loss-of-function mutations of CTNND2 target a neural-specific gene and are associated with transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 as the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate Stat3 signaling and confer mitogen independency and sensitivity to EGFR inhibition. These results provide important insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.
BCL6 encodes a transcription factor that represses genes necessary for the terminal differentiation of lymphocytes within germinal centers, and the misregulated expression of this factor is strongly implicated in several types of B cell lymphoma. The homodimeric BTB domain of BCL6 (also known as the POZ domain) is required for the repression activity of the protein and interacts directly with the SMRT and N-CoR corepressors that are found within large multiprotein histone deacetylase-containing complexes. We have identified a 17 residue fragment from SMRT that binds to the BCL6 BTB domain, and determined the crystal structure of the complex to 2.2 A. Two SMRT fragments bind symmetrically to the BCL6 BTB homodimer and, in combination with biochemical and in vivo data, the structure provides insight into the basis of transcriptional repression by this critical B cell lymphoma protein.
The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl--D-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel -barrel preceded by an amphipathic ␣ helix. The -barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual -strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.
BTB domain proteins An analysis of the protein architecture, genomic distribution and sequence conservation of BTB domain proteins in 17 fully sequenced eukaryotes reveals a high structural conservation and adaptation to different modes of self-association and interactions with non-BTB proteins.
The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.
Summary 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 non-toxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.
The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5-10% of C 2 H 2 -type zinc-finger transcription factors, as well as in some actinassociated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the fulllength PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surfaceexposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.The BTB domain (Broad-Complex, Tramtrack, and Bric à brac) (1, 2), also known as POZ (poxvirus and zinc finger) (3), is an evolutionarily conserved protein-protein interaction domain often found in developmentally regulated transcription factors. The domain is strongly implicated in the regulation of gene expression through the local control of chromatin conformation (4). The domain was first identified in a set of Drosophila and poxvirus genes (5), and examples of BTB domain genes have since been found in organisms ranging from yeast to man.A search of the current publicly available sequence databases reveals 56 distinct human BTB entries, of which 22 correspond to named, full-length genes, whereas the remaining entries are known only as tentative human consensus (THC) sequences, or expressed sequence tags (EST), (a tabulation of these genes can be found at http:͞͞xtal.oci.utoronto.ca͞prive͞ btbtable.html). Approximately two-thirds of the full-length human BTB genes also encode C 2 H 2 zinc finger modules, whereas approximately one-half of the remaining entries contain the kelch motif (4, 6). None of the known human BTB genes contain more than one single copy of the domain, making it likely that most, if not all, of the known BTB tentative human consensus (THC) and expressed sequence tag (EST) entries correspond to distinct genes. Based on the projection that there are 300-700 zinc finger proteins in man (7), we estimate that 5-10% of zinc finger proteins also contain BTB domains. The domain is known to form h...
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