Medicine and Health Sciences Commons^™

An Insulator Blocks Access To Enhancers By An Illegitimate Promoter, Preventing Repression By Transcriptional Interference., Miki Fujioka, Anastasiya Nezdyur, James B. Jaynes

Department of Biochemistry and Molecular Biology Faculty Papers

Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even …

Three-Dimensional Structure Of Human Cyclooxygenase (Hcox)-1., Morena Miciaccia, Benny Danilo Belviso, Mariaclara Iaselli, Gino Cingolani, Savina Ferorelli, Marianna Cappellari, Paola Loguercio Polosa, Maria Grazia Perrone, Rocco Caliandro, Antonio Scilimati

Department of Biochemistry and Molecular Biology Faculty Papers

The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 (hCOX-1) that we refined …

Structural Basis For The Homotypic Fusion Of Chlamydial Inclusions By The Snare-Like Protein Inca., Gino Cingolani, Michael Mccauley, Anna Lobley, Alexander J Bryer, Jordan Wesolowski, Deanna L Greco, Ravi K Lokareddy, Erik Ronzone, Juan R Perilla, Fabienne Paumet

Department of Biochemistry and Molecular Biology Faculty Papers

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during …

Post-Transcriptional Modifications To Trna--A Response To The Genetic Code Degeneracy., Ya-Ming Hou, Wei Yang

Department of Biochemistry and Molecular Biology Faculty Papers

No abstract provided.

G Protein Βγ Subunits Regulate Cardiomyocyte Hypertrophy Through A Perinuclear Golgi Phosphatidylinositol 4-Phosphate Hydrolysis Pathway., S Malik, R G Derubio, M Trembley, R Irannejad, Philip B Wedegaertner, A V Smrcka

Department of Biochemistry and Molecular Biology Faculty Papers

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein βγ subunits. Gβγ inhibition blocked ET-1-stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gβγ at the Golgi inhibited ET-1-dependent PI4P depletion and nuclear PKD activation. Translocation of Gβγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. …

Oligomerization, Conformational Stability And Thermal Unfolding Of Harpin, Hrpzpss And Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-Hrpzpss., Pradip K Tarafdar, Lakshmi Vasudev Vedantam, Rajeshwer S Sankhala, Pallinti Purushotham, Appa Rao Podile, Musti J Swamy

Department of Biochemistry and Molecular Biology Faculty Papers

HrpZ-a harpin from Pseudomonas syringae-is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both …

The Regulator Of G Protein Signaling (Rgs) Domain Of G Protein-Coupled Receptor Kinase 5 (Grk5) Regulates Plasma Membrane Localization And Function., Hua Xu, Xiaoshan Jiang, Ke Shen, Christopher C. Fischer, Philip B Wedegaertner

Department of Biochemistry and Molecular Biology Faculty Papers

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of …

Amino Acid-Dependent Stability Of The Acyl Linkage In Aminoacyl-Trna., Jacob R Peacock, Ryan R Walvoord, Angela Y Chang, Marisa C Kozlowski, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3'-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is …

Regulation Of Cell Death By Transfer Rna., Ya-Ming Hou, Xiaolu Yang

Department of Biochemistry and Molecular Biology Faculty Papers

SIGNIFICANCE: Both transfer RNA (tRNA) and cytochrome c are essential molecules for the survival of cells. tRNA decodes mRNA codons into amino-acid-building blocks in protein in all organisms, whereas cytochrome c functions in the electron transport chain that powers ATP synthesis in mitochondrion-containing eukaryotes. Additionally, in vertebrates, cytochrome c that is released from mitochondria is a potent inducer of apoptosis, activating apoptotic proteins (caspases) in the cytoplasm to dismantle cells. A better understanding of both tRNA and cytochrome c is essential for an insight into the regulation of cell life and death.

RECENT ADVANCES: A recent study showed that the …

Ash2 Acts As An Ecdysone Receptor Coactivator By Stabilizing The Histone Methyltransferase Trr., Albert Carbonell, Alexander Mazo, Florenci Serras, Montserrat Corominas

Department of Biochemistry and Molecular Biology Faculty Papers

The molting hormone ecdysone triggers chromatin changes via histone modifications that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with transcriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdysone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr …

Possible Steps Of Complete Disassembly Of Post-Termination Complex By Yeast Eef3 Deduced From Inhibition By Translocation Inhibitors., Shinya Kurata, Ben Shen, Jun O Liu, Nono Takeuchi, Akira Kaji, Hideko Kaji

Department of Biochemistry and Molecular Biology Faculty Papers

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions …

Testosterone Treatment Fails To Accelerate Disease In A Transgenic Mouse Model Of Spinal And Bulbar Muscular Atrophy., Erica S Chevalier-Larsen, Diane E Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Evidence from multiple animal models demonstrates that testosterone plays a crucial role in the progression of symptoms in spinal and bulbar muscular atrophy (SBMA), a condition that results in neurodegeneration and muscle atrophy in affected men. Mice bearing a transgene encoding a human androgen receptor (AR) that contains a stretch of 112 glutamines (expanded polyglutamine tract; AR112Q mice) reproduce several aspects of the human disease. We treated transgenic male AR112Q mice with testosterone for 6 months. Surprisingly, testosterone treatment of AR112Q males did not exacerbate the disease. Although transgenic AR112Q males exhibited functional deficits when compared with non-transgenics, long-term testosterone …

Regulation Of Energy Stores And Feeding By Neuronal And Peripheral Creb Activity In Drosophila., Koichi Iijima, Lijuan Zhao, Christopher Shenton, Kanae Iijima-Ando

Department of Biochemistry and Molecular Biology Faculty Papers

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores …

Multiple Domains In Siz Sumo Ligases Contribute To Substrate Selectivity., Alison Reindle, Irina Belichenko, Gwendolyn R Bylebyl, Xiaole L Chen, Nishant Gandhi, Erica S Johnson

Department of Biochemistry and Molecular Biology Faculty Papers

Saccharomyces cerevisiae contains two Siz/PIAS SUMO E3 ligases, Siz1 and Siz2/Nfi1, and one other known ligase, Mms21. Although ubiquitin ligases are highly substrate-specific, the degree to which SUMO ligases target distinct sets of substrates is unknown. Here we show that although Siz1 and Siz2 each have unique substrates in vivo, sumoylation of many substrates can be stimulated by either protein. Furthermore, in the absence of both Siz proteins, many of the same substrates are still sumoylated at low levels. Some of this residual sumoylation depends on MMS21. Siz1 targets its unique substrates through at least two distinct domains. Sumoylation of …