Medical Molecular Biology Commons^™

Three-Dimensional Context Rather Than Nls Amino Acid Sequence Determines Importin Α Subtype Specificity For Rcc1., Rajeshwer S. Sankhala, Ravi K. Lokareddy, Salma Begum, Ruth A. Pumroy, Richard E. Gillilan, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Active nuclear import of Ran exchange factor RCC1 is mediated by importin α3. This pathway is essential to generate a gradient of RanGTP on chromatin that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Here we identify the mechanisms of importin α3 selectivity for RCC1. We find this isoform binds RCC1 with one order of magnitude higher affinity than the generic importin α1, although the two isoforms share an identical NLS-binding groove. Importin α3 uses its greater conformational flexibility to wedge the RCC1 β-propeller flanking the NLS against its lateral surface, preventing steric clashes with its Armadillo-core. Removing …

Xenobiotic-Induced Activation Of Human Aryl Hydrocarbon Receptor Target Genes In Drosophila Is Mediated By The Epigenetic Chromatin Modifiers, Angelina A. Akishina, J. E. Vorontsova, Roman O. Cherezov, Ilja B. Mertsalov, Olga G. Zatsepina, Mikhail S. Slezinger, Vladislav M. Panin, Svetlana Petruk, Grigori N. Enikolopov, Alexander M. Mazo, Olga B. Simonova, Boris A. Kuzin

Department of Biochemistry and Molecular Biology Faculty Papers

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of …

Complex Interplay Of Kinetic Factors Governs The Synergistic Properties Of Hiv-1 Entry Inhibitors., Koree W. Ahn, Michael J. Root

Department of Biochemistry and Molecular Biology Faculty Papers

The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env-receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by …

Additional Sex Combs Interacts With Enhancer Of Zeste And Trithorax And Modulates Levels Of Trimethylation On Histone H3k4 And H3k27 During Transcription Of Hsp70., Taosui Li, Jacob W Hodgson, Svetlana Petruk, Alexander Mazo, Hugh W Brock

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: Maintenance of cell fate determination requires the Polycomb group for repression; the trithorax group for gene activation; and the enhancer of trithorax and Polycomb (ETP) group for both repression and activation. Additional sex combs (Asx) is a genetically identified ETP for the Hox loci, but the molecular basis of its dual function is unclear.

RESULTS: We show that in vitro, Asx binds directly to the SET domains of the histone methyltransferases (HMT) enhancer of zeste [E(z)] (H3K27me3) and Trx (H3K4me3) through a bipartite interaction site separated by 846 amino acid residues. In Drosophila S2 cell nuclei, Asx interacts with …

The 11s Proteasomal Activator Regγ Impacts Polyglutamine-Expanded Androgen Receptor Aggregation And Motor Neuron Viability Through Distinct Mechanisms., Jill M. Yersak, Heather L. Montie, Erica S. Chevalier-Larsen, Yuhong Liu, Lan Huang, Martin Rechsteiner, Diane E. Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is caused by expression of a polyglutamine (polyQ)-expanded androgen receptor (AR). The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ) is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28β) counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation …

Structure Activity Relationship Studies Of Novel Diarylpentanoid Analogs Targeting The Androgen Receptor In Prostate Cancer Cells, Haili Coffin, Marco Bisoffi

Student Scholar Symposium Abstracts and Posters

The development of prostate cancer (PCa) relies strongly on the activation of the androgen receptor (AR) signaling pathway by its natural ligand dihydrotestosterone. Furthermore, PCa progression to metastatic disease represents oncogene addiction to AR activity. Androgen ablation therapy is thus a mainstay therapy against this disease, but the development of ligand-independent AR activation and persisting AR expression eventually leads to castration resistant PCa (CRPC). Therefore, down-regulation of AR expression in PCa cells may be an effective therapeutic modality. The diarylpentanoid ca27 has previously been shown to down-regulate AR expression by an unknown mechanism of action. The present work represents a …

Foxo3 Increases Mir-34a To Cause Palmitate-Induced Cholangiocyte Lipoapoptosis., Sathish Kumar Natarajan, Bailey A. Stringham, Ashley M. Mohr, Cody J. Wehrkamp, Sizhao Lu, Mary A. Smith, Dee Harrison-Findik, Justin L. Mott

Journal Articles: Biochemistry & Molecular Biology

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. …

Kinetic Studies Of Dna Repair Enzyme Alkbh2, Michael R. Vittori

Senior Honors Projects

The genomes of living organisms are under constant bombardment from various sources, including chemical modification stemming from processes within the organisms themselves or from exogenous agents, and from radiation. These sources of genomic damage may induce structural changes in the genome’s most basic functional units, the nucleotides that comprise DNA. Damage to an organism’s DNA may result in the production of dysfunctional or nonfunctional proteins. Failure to repair such damage may result in the compounding of successive mutations within the organism’s genome, the pathogenesis of cancer and various genetic disorders in humans. To ensure their viability, organisms have developed unique …

Chemical And Structural Characterization Of A Model Post-Termination Complex (Potc) For The Ribosome Recycling Reaction: Evidence For The Release Of The Mrna By Rrf And Ef-G., Nobuhiro Iwakura, Takeshi Yokoyama, Fabio Quaglia, Kaoru Mitsuoka, Kazuhiro Mio, Hideki Shigematsu, Mikako Shirouzu, Akira Kaji, Hideko Kaji

Department of Biochemistry and Molecular Biology Faculty Papers

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we …

Role Of Dendritic Cells In Pathology Of Respiratory Syncytial Virus Infection In Neonates, Bishwas Shrestha

Theses and Dissertations (ETD)

Respiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis in children. We have shown that neonatal mice respond to primary RSV infection with T helper type 2 (Th2) biased immune responses, which are enhanced following reinfection. Dendritic cells (DCs) including myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) play important roles in driving host responses to RSV infection. mDCs present antigens to help Th cells differentiate, and pDCs protect against viral infection through type I interferons (IFNs). Despite data demonstrating importance of mDCs and pDCs in protection against RSV, it has not been studied in an age appropriate …

Dysregulated Gpcr Signaling And Therapeutic Options In Uveal Melanoma., Vivian Chua, Dominic Lapadula, Clinita Randolph, Jeffrey L. Benovic, Philip B. Wedegaertner, Andrew E. Aplin

Department of Biochemistry and Molecular Biology Faculty Papers

Uveal melanoma is the most common primary intraocular malignant tumor in adults and arises from the transformation of melanocytes in the uveal tract. Even after treatment of the primary tumor, up to 50% of patients succumb to metastatic disease. The liver is the predominant organ of metastasis. There is an important need to provide effective treatment options for advanced stage uveal melanoma. To provide the preclinical basis for new treatments, it is important to understand the molecular underpinnings of the disease. Recent genomic studies have shown that mutations within components of G protein-coupled receptor (GPCR) signaling are early events associated …

Determining The Protective Effects Of Quercetin Against Cadmium Toxicity In Human Embryonic Kidney Cells, Caroline N. Smith

Undergraduate Theses

Cadmium is a toxic industrial and environmental pollutant found in groundwater, air, soils, food and cigarettes. Chronic intake of low levels of cadmium has been shown to result in renal dysfunction due to cell death which can occur via apoptosis as well as necrosis. Previous studies have shown that plant extracts containing quercetin, a flavonoid found in many fruits and vegetables, protect against cadmium toxicity in rat liver hepatocytes. To determine if quercetin may have a protective effect in a cadmium-treated human embryonic kidney cell line, HEK-293 cells were treated using concentrations of cadmium chloride from 10 to 50 μM …

A Genetically Encoded Fluorescent Trna Is Active In Live-Cell Protein Synthesis., Isao Masuda, Takao Igarashi, Reiko Sakaguchi, Ram G. Nitharwal, Ryuichi Takase, Kyu Young Han, Benjamin J. Leslie, Cuiping Liu, Howard Gamper, Taekjip Ha, Suparna Sanyal, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular …

Structural And Functional Analysis Of A Β2-Adrenergic Receptor Complex With Grk5., Konstantin E. Komolov, Yang Du, Nguyen Minh Duc, Robin M. Betz, João P.G.L.M. Rodrigues, Ryan D. Leib, Dhabaleswar Patra, Georgios Skiniotis, Christopher M. Adams, Ron O. Dror, Ka Young Chung, Brian K. Kobilka, Jeffrey L. Benovic

Department of Biochemistry and Molecular Biology Faculty Papers

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. …

Portal Protein Functions Akin To A Dna-Sensor That Couples Genome-Packaging To Icosahedral Capsid Maturation., Ravi K. Lokareddy, Rajeshwer S. Sankhala, Ankoor Roy, Pavel V. Afonine, Tina Motwani, Carolyn M M. Teschke, Kristin N. Parent, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or 'procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity …

Urokinase Plasminogen Activator Expression Is Regulated By P53 Harboring The Lung Cancer-Specific Mutation V157f, Julie Barta, Md, Kristen Pauley, Steven B. Mcmahon, Phd

Department of Biochemistry and Molecular Biology Posters

OBJECTIVES

• To define the mutant p53-regulated transcriptome of lung cancer cells with alterations at V157 and R158 in the p53 tumor suppressor.
• To determine the biological effects of lung-enriched p53 mutations in lung cancer cells.

Cleavage Of Dfna5 By Caspase-3 During Apoptosis Mediates Progression To Secondary Necrotic/Pyroptotic Cell Death., Corey Rogers, Teresa Fernandes-Alnemri, Lindsey Mayes, Diana Alnemri, Gino Cingolani, Emad S. Alnemri

Department of Biochemistry and Molecular Biology Faculty Papers

Apoptosis is a genetically regulated cell suicide programme mediated by activation of the effector caspases 3, 6 and 7. If apoptotic cells are not scavenged, they progress to a lytic and inflammatory phase called secondary necrosis. The mechanism by which this occurs is unknown. Here we show that caspase-3 cleaves the GSDMD-related protein DFNA5 after Asp270 to generate a necrotic DFNA5-N fragment that targets the plasma membrane to induce secondary necrosis/pyroptosis. Cells that express DFNA5 progress to secondary necrosis, when stimulated with apoptotic triggers such as etoposide or vesicular stomatitis virus infection, but disassemble into small apoptotic bodies when DFNA5 …

Multifaceted Role Of Neuropilins In The Immune System: Potential Targets For Immunotherapy., Sohini Roy, Arup K. Bag, Rakesh Singh, James E. Talmadge, Surinder K. Batra, Kaustubh Datta

Journal Articles: Biochemistry & Molecular Biology

Neuropilins (NRPs) are non-tyrosine kinase cell surface glycoproteins expressed in all vertebrates and widely conserved across species. The two isoforms, such as neuropilin-1 (NRP1) and neuropilin-2 (NRP2), mainly act as coreceptors for class III Semaphorins and for members of the vascular endothelial growth factor family of molecules and are widely known for their role in a wide array of physiological processes, such as cardiovascular, neuronal development and patterning, angiogenesis, lymphangiogenesis, as well as various clinical disorders. Intriguingly, additional roles for NRPs occur with myeloid and lymphoid cells, in normal physiological as well as different pathological conditions, including cancer, immunological disorders, …