Browsing by Subject "DNA-Binding Proteins"
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Item Biochemical and Genetic Studies on CC2D1A, a new NF-κB Activator and a Regulator of Synaptic Functions(2010-05-14) Zhao, Meng; Chen, ZhijianCC2D1A is an evolutionarily conserved gene from worm to human. It belongs to a new protein family with four DM14 domains at the NH2 terminus and a C2 domain at the COOH terminus. The function of this protein family remains largely unknown. CC2D1A has been identified as a new NF-κB activator through a large scale screen of human genes by Matsuda et al. Here I show that the conserved DM14 and C2 domains of CC2D1A are important for NF-κB activation. CC2D1A activates the IKK complex and NF-κB target genes through several key components in the canonical pathway including ubiquitin-conjugating enzyme UBC13, a RING domain ubiquitin ligase TRAF2, a protein kinase TAK1, and an essential regulator of IKK complex, NEMO. CYLD, a deubiquitination enzyme specific for Lysine-63 linked polyubiquitin chains, negatively regulates the activity of CC2D1A. These results suggest that CC2D1A activates NF-κB through the canonical IKK pathway. In an attempt to identify the physiological function of CC2D1A, I generated CC2D1A knockout mice. The KO animals die right after birth apparently due to their inability to breathe. Histological analysis identified no significant anatomical defects. In particular, brain, heart and muscle are normal with regard to morphology. In addition, neuromuscular junction at the diaphragm is formed in the absence of Cc2d1a. Human patients with mutations in the gene suffer from mental retardation, implying that Cc2d1a functions in the central nervous system (CNS). Here I show that Cc2d1a expression is enriched in the brain. Deletion of Cc2d1a impairs synapse maturation and function in cortical neurons. Our study may help understand the molecular basis of some human diseases such as mental retardation.Item KU70 Binding Protein 5 (KUB5), A Novel Factor in DNA Double Strand Break Repair and Radio-Resistance in Human Breast Cancer(2011-02-01T19:37:12Z) Rommel, Amy Ann; Boothman, David A.DNA double strand breaks (DSBs) are considered both mutagenic and carcinogenic if left un-repaired resulting in genomic instability and ultimately cancer. There are two main pathways for DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). Defects in DSB repair have already been associated with breast cancer formation and increased breast cancer risk. Breast cancer susceptibility genes, BRCA1 and BRCA2 are largely thought to be involved with HR while LIG4, XRCC4, and Ku70 are linked to NHEJ. Deficiencies in any one of these genes can predispose individuals to breast cancer. In addition to predisposition to breast cancer, altered DNA repair processes can influence chemo- and radiotherapy efficacy by creating resistance to therapy. To study NHEJ further, our laboratory has identified a novel Ku70 binding protein #5 (KUB5) by a yeast two-hybrid screen using Ku70 as bait. Loss of RTT103, a putative yeast homolog of KUB5, resulted in increased sensitivity to IR, similar to that observed in hdf1-deletion yeast, the yeast homolog of Ku70. Results also show that RTT103-deletion yeast are deficient in repairing blunt and non-compatible DNA ends and re-expression of hKub5 can correct the IR-sensitivity and DNA repair deficiency of these deficient yeast demonstrating a strong functional model for human KUB5 function in yeast. Analyses of breast cancer cell lines for their KUB5 protein expression yielded a strong correlation between KUB5 protein level and sensitivity to DNA damage. These data strongly suggests that KUB5 is a novel repair factor involved in NHEJ and endogenous over-expression of KUB5 plays a role in chemotherapeutic and/or radio-therapeutic resistance via increasing the capacity to facilitate NHEJ repair of DSBs in breast cancer cells.Item Multiple Approaches to Study of Steroidogenic Factor 1 : Identification of a Novel Regulatory Element and Identification of Novel Target Genes(2005-05-03) Stallings, Nancy Ruth; Parker, Keith L.Steroidogenic Factor 1 (SF-1) is an essential component of the hypothalamic-pituitary-adrenal-gonadal axis. SF-1 knockout (KO) mice lack adrenals, gonads, the ventromedial hypothalamic nucleus (VMH), and pituitary gonadotropes. SF-1 is a transcription factor implicated in the regulation of many genes important in endocrine function. Research into the regulation of SF-1 expression, mostly focused on the proximal promoter, has been unable to fully explain the expression pattern of SF-1. I used DNase hypersensitivity mapping to search for novel regulatory regions of the SF-1 genomic region. One region between the 6th and 7th exons of SF-1 had tissue specific DNase I hypersensitivity. Analysis of this region revealed high conservation with the human genomic sequence and a smaller region that was also highly conserved in the chicken genomic sequence. Transient transfection assays and electrophoretic mobility shift assays have been employed to investigate this conserved element for enhancer activity. Numerous genes are target genes of SF-1, yet loss of known target genes fail to explain why the adrenals, gonads and VMH fail to develop in SF-1 KO mice. I used an SF-1/eGFP transgene as a reporter in both WT and KO E16.5 embryos. eGFP+ cells from the developing VMH of these mice were collected through the use of FACS. Several potential target genes of SF-1 have been identified and analysis of these genes is an ongoing project.Item Regulatory Mechanism of the RNAi Pathway(2011-12-14) Liu, Ying; Liu, QinghuaRNA interference (RNAi) is post-transcriptional gene silencing initiated by Dicer, a RNase III that processes double-stranded RNA (dsRNA) precursors into small interfering RNA (siRNA). In Drosophila, Dicer2 and R2D2 coordinately recruit duplex siRNA to the effector RNA-induced silencing complex (RISC), wherein single-stranded siRNA guides the endoribonuclease Argonaute (Ago) to catalyze sequence-specific cleavage of complementary mRNA. It remains unclear as to what constitutes holo-RISC, how is RISC assembled and how is RISC regulated. Here we took a candidate approach to reconstitute for the first time the long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We further employed this core reconstitution system to purify a RNAi regulator that we named C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a novel Mg2+ -dependent endoribonuclease that promotes RISC activation by removing the siRNA passenger strand cleavage products. Similar as Drosophila C3PO, human C3PO also degrades passenger strand fragments and facilitates RISC activation. RISC is a multiple-turnover enzyme, wherein single-stranded (ss)-siRNA guides Ago2 to catalyze sequence-specific cleavage of the target mRNA at the effector step. We employed human minimal RISC reconstitution system to purify antoantigen La as a novel activator of the RISC effector step. Biochemical studies indicated that La promotes the multiple-turnover of RISC catalysis by facilitating the release of RISC cleaved products. Moreover, we demonstrated that La is required for efficient RNAi, antiviral defense, and transposon silencing in mammalian and Drosophila cells. Taken together, our findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. The robust reconstitution system establishes a powerful platform for in-depth studies of the assembly, function, and regulation of RISC. Similar to the discovery of C3PO and La, it can be used to identify novel regulators and study post-translational regulations of RNAi, therefore, connecting RNAi to other cellular signaling pathways. As such, these biomedical studies could have a major and lasting impact on the biological understanding and therapeutic application of RNAi.Item Role of c-Cbl in Invasion of Mammalian Cells by Rickettsia Conorii(2007-08-08) Ravikumar, Sai P.; UnknownSpecific AimsRickettsia conorii is an intracellular bacterium that causes Mediterranean spotted fever. R. conorii is transmitted from ticks to humans and invades vascular endothelial cells. A recent publication (Martinez et al., 2005) has identified Ku70 as a host cell receptor that binds to rOmpB, an R. conorii surface ligand. The engagement of Ku70 by rOmpB leads to a rapid ubiquitination of Ku70 by c-Cbl, followed by the entry of R. conorii into the host cell. However, the role of c-Cbl in ubiquitinating Ku70 and communicating with the invading bacterium remain to be clarified. Based on the report of Martinez et al and other groups on the roles played by R. conorii surface ligands rOmpA and rOmpB, we propose the following hypothesis: Hypothesisc-Cbl ubiquitinates Ku70 in response to signals from a Rickettsia conorii cell surface protein called rOmpA. This ubiquitination event enables Ku70 and the attached bacteria to be endocytosed into the cell. Specific Aims1. To analyse the effect of c-Cbl - mediated ubiquitination on Ku70 localisation. We will investigate if ubiquitination of Ku70 by c-Cbl is necessary for Ku70 to localize to the bacterial entry foci. We will culture c-Cbl deficient cells and observe the effects of R. conorii infection on them.2. To map the putative signal(s) involved in the communication between R. conorii rOmpA outside the cell and c-Cbl inside the cell. We will identify the putative rOmpA receptor on host cells and protein(s) that may act as bridges of communication between rOmpA and c-Cbl.3. To investigate the mechanism of how ubiquitination of Ku70 enables R. conorii to enter a host cell. We will determine if any c-Cbl interacting proteins and/or endocytic proteins are recruited to the cell surface by ubiquitinated Ku70 to form a phagosome. If endocytic proteins are recruited, we will proceed.Item Steroidogenic Factor 1 and Beta-Catenin: Two Critical Regulators of Endocrine Organ Development(2009-06-18) Reuter, Anne Louise; Repa, Joyce J.The endocrine system comprises a diverse array of organs and hormones that regulate many aspects of development and homeostasis. Steroidogenic hormones - secreted by the adrenal cortex, testis, and ovary - are required for electrolyte balance, maintenance of intermediary metabolism, and reproduction. The nuclear receptor steroidogenic factor 1 (SF-1, officially designated NR5A1) originally was identified as a transcriptional regulator of steroidogenic synthetic enzymes. In addition to expression in the adrenal cortex and somatic cells of the gonads, however, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and pituitary gonadotropes, suggesting a broader role in endocrine physiology. Global knockout of SF-1 in mice confirmed this possibility, as it resulted in complete adrenal and gonadal agenesis and XY sex reversal, causing postnatal death due to adrenal insufficiency. Humans with mutations in SF-1 exhibit a spectrum of phenotypes ranging from 46, XY sex reversal and adrenal insufficiency to patients with normal adrenal function and mild gonadal dysgenesis. Herein is reported a patient with compound heterozygosity for a previously described SF-1 polymorphism and a novel mutation, p.R84C, which impairs DNA binding and transactivation activity. SF-1 interacts with numerous coactivators, including beta-catenin, a central mediator of the canonical Wnt signaling pathway. Upon activation by Wnt, beta-catenin translocates to the nucleus where it enhances the transcription of Wnt target genes, and - as recently appreciated - a subset of SF-1 gene targets. Due to the reported functional synergy between SF-1 and beta-catenin, and as Wnt4 deficiency in mice resulted in adrenal and gonadal defects, we investigated the consequences of beta-catenin disruption in certain SF-1-expressing tissues, specifically the adrenal and pituitary glands, and the VMH. SF-1/Cre-mediated beta-catenin knockout mice died immediately after birth and lacked adrenal glands. In contrast, the VMH and pituitary gland were largely unaffected from a structural viewpoint. Analysis of adrenal development revealed that though the adrenal primordium forms, adrenocortical cell numbers quickly decline due to impaired proliferation. The potential for beta-catenin/SF-1 synergy on a fetal adrenal specific enhancer was investigated. These results implicate beta-catenin - presumably as part of the Wnt signaling pathway - as a required factor in adrenocortical development.Item Structural Basis for Signal Transduction in LOV Blue Light Photoreceptors(2011-08-26T17:33:55Z) Nash, Abigail I.; Gardner, Kevin H.This research focused on studying the mechanisms of signal transduction within Light-Oxygen-Voltage domains, a subset of the PAS domain family. The first of two projects addressed intradomain signaling from the hydrophobic core to the domain surface. In this study, we addressed the role of a specific conserved residue in transmitting activating signal from the domain core to surface structural elements. Through biophysical and biochemical studies of LOV proteins containing point mutations at key residues, we determined that structural strain placed on the domain following light-induced covalent adduct formation regulates both structural based signal transduction as well as dark state recovery kinetics. In the second project, I characterized a novel LOV containing protein comprised of an N-terminal LOV domain and a C-terminal DNA binding helix-turn-helix (HTH) domain. Following initial characterization of this protein, I was able to determine how light-induced covalent adduct formation in the N-terminus leads to interdomain separation through release of inhibitory contacts with the HTH domain, allowing for DNA binding. Comparisons of this protein with other known HTH proteins highlight the conserved signal transduction pathways of both the LOV domain and the HTH domain.Item Studies Of Smoothened In Hedgehog Signaling Pathway(2006-12-20) Tong, Chao; Jiang, JinThe Hedgehog (Hh) family of morphogens controls cell growth and patterning in both vertebrates and invertebrates. Malfunction of Hh signaling has been implicated in numerous human disorders. As the Hh signal transducer, the seven-transmembrane protein Smoothened (Smo) is highly regulated. It is still a mystery how Smo transduces graded Hh signal to downstream components. Although Smo shares some structural similarity with G protein coupled receptors (GPCR), there is little evidence that G proteins are involved in Hh signal transduction in physiological settings. A kinesin like protein Costal2 (Cos2) and a serine/threonine kinase Fused (Fu) form complexes with the transcription factor Cubitus-interrupts (Ci), which is essential for Hh signal transduction. However, how Smo transduces Hh signal to this complex is still not clear. In this study, we found that Smo interacts with Cos2-Fu complex through its C-terminal tail, which is essential for the Hh pathway activation. In response to Hh, Smo is phosphorylated and accumulated on the cell surface. However, the kinases responsible for Hh induced Smo phosphorylation are still unknown. It is also not clear whether phosphorylation regulates Smo activity or not. In this study, I found that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell surface accumulation and activity in response to Hh. PKA and CKI phosphorylate Smo directly at multiple sites which form three clusters in Smo C-terminal tail. In cooperation with Jianhang, we found that phosphorylation deficient forms of Smo failed to accumulate on the cell surface and were unable to transduce Hh signal. By contrast, phosphorylation mimicking forms of Smo have increased cell surface accumulation and constitutive activity. In addition, we also found the levels of Smo cell surface accumulation and activity correlate with its phosphorylation levels, suggesting that the graded Smo activity may be regulated by differential phosphorylation of its C-terminal tail. Furthermore, I have identified multiple Arginine clusters in Smo the C-terminal tail that negatively regulate Smo activity by preventing Smo cell surface accumulation and keeping Smo C-terminal tail in a closed inactive conformation maintained by intramolecular electrostatic interactions. I have also found that the number of arginine clusters is reversely correlated with Smo cell-surface expression and activity. I also provided evidence that phosphorylation antagonizes the negative effects of the Arginines by neutralizing the positive charges they carry, which lets Smo C-terminal tail adopts an open and active conformation and promotes Smo cell surface accumulation. Based on these data, we proposed that multiple arginine clusters provide a way to finetune Smo activity in response to different Hh levels by differentially phosphorylating Smo C-terminal tail. This study also showed that Gprk2, a G protein coupled receptor kinase (GRK), plays a positive role in regulating Hh signalling. I provided evidence that Gprk2 interacts with Smo Ctail. Furthermore, I identified a new CKI phosphorylation cluster that appears to be critical for Smo endocytosis and activation.Item TDP-43 Is Directed to Stress Granules by Sorbitol, a Novel Physiological Osmotic and Oxidative Stressor(2012-07-20) Dewey, Colleen Marie; Yu, GangTDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and post-transcriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify TDP-43 association with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules and incorporates into stress granules more early; in striking contrast, wild-type TDP-43 forms more stress granules over time, but granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies. [Keywords: TDP-43, FTLD-U, ALS, TARDBP, stress granules, sorbitol, neurodegeneration, RNA-binding proteins]