IU Indianapolis ScholarWorks :: Browsing by Subject "DNA-binding proteins"

Browsing by Subject "DNA-binding proteins"

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ARID3B Induces Tumor Necrosis Factor Alpha Mediated Apoptosis While a Novel ARID3B Splice Form Does Not Induce Cell Death
(Public Library of Science, 2012) Joseph, Stancy; Deneke, Victoria E.; Cowden Dahl, Karen D.; Medicine, School of Medicine
Alternative splicing is a common occurrence in many cancers. Alternative splicing is linked with decreased apoptosis and chemoresistance in cancer cells. We previously demonstrated that ARID3B, a member of the AT-rich interactive domain (ARID) family of DNA binding proteins, is overexpressed in ovarian cancer. Therefore we wanted to assess the effect of ARID3B splice forms on cell viability. We identified a novel splice form of the ARID3B gene (designated as ARID3B Sh), which lacks the C-terminal exons 5-9 present in the full-length isoform (ARID3B Fl). ARID3B Fl is expressed in a variety of cancer cell lines. Expression of ARID3B Sh varied by cell type, but was highly expressed in most ovarian cancer lines. ARID3B is modestly transcriptionally activated by epidermal growth factor receptor (EGFR) signaling through the PEA3 transcription factor. We further found that ARID3B Fl is predominantly nuclear but is also present at the plasma membrane and in the cytosol. Endogenous ARID3B Sh is present in nuclear fractions, yet, when overexpressed ARID3B Sh accumulates in the cytosol and membrane fractions. The differential localization of these isoforms suggests they have different functions. Importantly, ARID3B Fl overexpression results in upregulation of pro-apoptotic BIM and induces Tumor Necrosis Factor alpha (TNFα) and TNF-related apoptosis inducing ligand (TRAIL) induced cell death. The ARID3B Fl-induced genes include TNFα, TRAIL, TRADD, TNF-R2, Caspase 10 and Caspase 7. Interestingly, ARID3B Sh does not induce apoptosis or expression of these genes. ARID3B Fl induces death receptor mediated apoptosis while the novel splice form ARID3B Sh does not induce cell death. Therefore alternative splice forms of ARID3B may play different roles in ovarian cancer progression.
Bcl6 Controls the Th2 Inflammatory Activity of Regulatory T Cells by Repressing Gata3 Function
(Oxford University Press, 2012) Sawant, Deepali V.; Sehra, Sarita; Nguyen, Evelyn T.; Jadhav, Rohit; Englert, Kate; Shinnakasu, Ryo; Hangoc, Giao; Broxmeyer, Hal E.; Nakayama, Toshinori; Perumal, Narayanan B.; Kaplan, Mark H.; Dent, Alexander L.; Microbiology and Immunology, School of Medicine
The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.
CXXC finger protein 1 is important for hematopoiesis
(2005) Young, Suzanne R.L.
Deep top-down proteomics revealed significant proteoform-level differences between metastatic and nonmetastatic colorectal cancer cells
(American Association for the Advancement of Science, 2022) McCool, Elijah N.; Xu, Tian; Chen, Wenrong; Beller, Nicole C.; Nolan, Scott M.; Hummon, Amanda B.; Liu, Xiaowen; Sun, Liangliang; BioHealth Informatics, School of Informatics and Computing
Understanding cancer metastasis at the proteoform level is crucial for discovering previously unknown protein biomarkers for cancer diagnosis and drug development. We present the first top-down proteomics (TDP) study of a pair of isogenic human nonmetastatic and metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). We identified 23,622 proteoforms of 2332 proteins from the two cell lines, representing nearly fivefold improvement in the number of proteoform identifications (IDs) compared to previous TDP datasets of human cancer cells. We revealed substantial differences between the SW480 and SW620 cell lines regarding proteoform and single amino acid variant (SAAV) profiles. Quantitative TDP unveiled differentially expressed proteoforms between the two cell lines, and the corresponding genes had diversified functions and were closely related to cancer. Our study represents a pivotal advance in TDP toward the characterization of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.
Deleted in malignant brain tumor 1 genetic variation confers urinary tract infection risk in children and mice
(Wiley, 2021-07) Hains, David S.; Polley, Shamik; Liang, Dong; Saxena, Vijay; Arregui, Samuel; Ketz, John; Barr-Beare, Evan; Rawson, Ashley; Spencer, John D.; Cohen, Ariel; Hansen, Pernille L.; Tuttolomondo, Martina; Casella, Cinzia; Ditzel, Henrik J.; Cohen, Daniel; Hollox, Edward J.; Schwaderer, Andrew L.; Pediatrics, School of Medicine
Design, Synthesis and Study of DNA-Targeted Benzimidazole-Amino Acid Conjugates
(2013-07-12) Garner, Matthew L.; Long, Eric C. (Eric Charles); Minto, Robert; O'Donnell, Martin J.
The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.
Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells
(PLOS, 2021-12-23) Day, Richard N.; Day, Kathleen H.; Pavalko, Fredrick M.; Anatomy, Cell Biology and Physiology, School of Medicine
Earlier, we proposed the "mechanosome" concept as a testable model for understanding how mechanical stimuli detected by cell surface adhesion molecules are transmitted to modulate gene expression inside cells. Here, for the first time we document a putative mechanosome involving Src, Pyk2 and MBD2 in MLO-Y4 osteocytes with high spatial resolution using FRET-FLIM. Src-Pyk2 complexes were concentrated at the periphery of focal adhesions and the peri-nuclear region. Pyk2-MBD2 complexes were located primarily in the nucleus and peri-nuclear region. Lifetime measurements indicated that Src and MBD2 did not interact directly. Finally, mechanical stimulation by fluid flow induced apparent accumulation of Src-Pyk2 protein complexes in the peri-nuclear/nuclear region, consistent with the proposed behavior of a mechanosome in response to a mechanical stimulus.
Elucidating the role of redox effects and the KU80 C-Terminal region in the regulation of the human DNA repair protein KU
(2010-07-20T16:00:25Z) McNeil, Sara M.; Turchi, John J.; Harrington, Maureen A.; Georgiadis, Millie M.
DNA double strand breaks (DSB) are among the most lethal forms of DNA damage and can occur as a result of ionizing radiation (IR), radiomimetic agents, endogenous DNA-damaging agents, etc. If left unrepaired DSB’s can cause cell death, chromosome translocation and carcinogenesis. In humans, DSB are repaired predominantly by the non-homologous end joining (NHEJ) pathway. Ku, a heterodimer consisting of Ku70 and Ku80, functions in the recognition step of this pathway through binding DNA termini. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to create the full DNA-PK heterotrimer. Formation of DNA-PK results in autophosphorylation as well as phosphorylation of downstream proteins of the NHEJ pathway. Previous work shows that the extreme C-terminus of Ku80 stimulates the kinase activity of DNA-PKcs, and Ku DNA binding is regulated as a function of redox via stimulation of a conformational change when oxidized resulting in a decrease in DNA binding activity. To further understand these methods of regulation of Ku and DNA-PK, a pair of mutants has been constructed; one consisting of full length Ku70 and truncated Ku80 (Ku70/80ΔC) lacking 182 C-terminal amino acids. The removal of these amino acids was shown to have little to no effect on the proteins expression, stability or DNA binding, as determined by SDS-PAGE, western blot analysis and electrophoretic mobility shift assay (EMSA). When oxidized Ku70/80ΔC showed a decrease in DNA binding similar to that seen in wild type, however when re-reduced the mutant did not recover to the same extent as wild type. A second mutant was constructed, containing amino acids 590-732 of Ku80 (Ku80CTR), to further understand the mechanism by which Ku80 C-terminus interacts with the rest of the Ku heterodimer. Possible protein-protein interactions were evaluated by Ni-NTA affinity, gel filtration chromatography, fluorescence polarization and two forms of protein-protein cross-linking. Ni-NTA agarose affinity, and gel filtration chromatography failed to reveal an interaction in the presence or absence of DNA. However, photo-induced cross-linking of unmodified proteins (PICUP) as well as EDC cross-linking demonstrated an interaction which was not affected by DNA. The work presented here demonstrates that the interaction between Ku80CTR and Ku is rather weak, but it does exist and plays a relatively large role in the NHEJ pathway.
Exploring the mechanism of action of spore photoproduct lyase
(2014-08-27) Nelson, Renae; Li, Lei; Long, Eric C. (Eric Charles); McLeish, Michael J.
Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.
Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood
(Elsevier, 2017-08-03) Edvardson, Simon; Nicolae, Claudia M.; Agrawal, Pankaj B.; Mignot, Cyril; Payne, Katelyn; Prasad, Asuri Narayan; Prasad, Chitra; Sadler, Laurie; Nava, Caroline; Mullen, Thomas E.; Begtrup, Amber; Baskin, Berivan; Powis, Zöe; Shaag, Avraham; Keren, Boris; Moldovan, George-Lucian; Elpeleg, Orly; Pediatrics, School of Medicine
Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G>A in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5'- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.

Browsing by Subject "DNA-binding proteins"

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