Medicine and Health Sciences Commons^™

Structural Insights Into Rna Bridging Between Hiv-1 Vif And Antiviral Factor Apobec3g, Takahide Kouno, Satoshi Shibata, Megumi Shigematsu, Jaekyung Hyun, Tae Gyun Kim, Hiroshi Matsuo, Matthias Wolf

Computational Medicine Center Faculty Papers

Great effort has been devoted to discovering the basis of A3G-Vif interaction, the key event of HIV's counteraction mechanism to evade antiviral innate immune response. Here we show reconstitution of the A3G-Vif complex and subsequent A3G ubiquitination in vitro and report the cryo-EM structure of the A3G-Vif complex at 2.8 Å resolution using solubility-enhanced variants of A3G and Vif. We present an atomic model of the A3G-Vif interface, which assembles via known amino acid determinants. This assembly is not achieved by protein-protein interaction alone, but also involves RNA. The cryo-EM structure and in vitro ubiquitination assays identify an adenine/guanine base …

Starvation Sensing By Mycobacterial Rela/Spot Homologue Through Constitutive Surveillance Of Translation, Yunlong Li, Soneya Majumdar, Ryan Treen, Manjuli R. Sharma, Jamie Corro, Howard B. Gamper, Swati R. Manjari, Jerome Prusa, Nilesh K. Banavali, Christina L. Stallings, Ya-Ming Hou, Rajendra K. Agrawal, Anil K. Ojha

Department of Biochemistry and Molecular Biology Faculty Papers

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in …

Design And Preclinical Evaluation Of A Universal Sars-Cov-2 Mrna Vaccine, Jane Qin, Ju Hyeong Jeon, Jiangsheng Xu, Laura Katherine Langston, Ramesh Marasini, Stephanie Mou, Brian Montoya, Carolina R Melo-Silva, Hyo Jin Jeon, Tianyi Zhu, Luis J. Sigal, Renhuan Xu, Huabin Zhu

Department of Microbiology and Immunology Faculty Papers

Because of the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated …

Parp1 Associates With R-Loops To Promote Their Resolution And Genome Stability, Natalie Laspata, Parminder Kaur, Sofiane Yacine Mersaoui, Daniela Muoio, Zhiyan Silvia Liu, Maxwell Henry Bannister, Hai Dang Nguyen, Caroline Curry, John M. Pascal, Guy G. Poirier, Hong Wang, Jean-Yves Masson, Elise Fouquerel

Student Papers, Posters & Projects

PARP1 is a DNA-dependent ADP-Ribose transferase with ADP-ribosylation activity that is triggered by DNA breaks and non-B DNA structures to mediate their resolution. PARP1 was also recently identified as a component of the R-loop-associated protein-protein interaction network, suggesting a potential role for PARP1 in resolving this structure. R-loops are three-stranded nucleic acid structures that consist of a RNA-DNA hybrid and a displaced non-template DNA strand. R-loops are involved in crucial physiological processes but can also be a source of genome instability if persistently unresolved. In this study, we demonstrate that PARP1 binds R-loops in vitro and associates with R-loop formation …

Semi-Quantitative Detection Of Pseudouridine Modifications And Type I/Ii I/Ii Hypermodifications In Human Mrnas Using Direct Long-Read Sequencing, Sepideh Tavakoli, Mohammad Nabizadeh, Amr Makhamreh, Howard Gamper, Caroline A Mccormick, Neda K Rezapour, Ya-Ming Hou, Meni Wanunu, Sara H Rouhanifard

Department of Biochemistry and Molecular Biology Faculty Papers

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic …

Complexity Of Progranulin Mechanisms Of Action In Mesothelioma, Elisa Ventura, Christopher Xie, Simone Buraschi, Antonino Belfiore, Renato V. Iozzo, Antonio Giordano, Andrea Morrione

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background: Mesothelioma is an aggressive disease with limited therapeutic options. The growth factor progranulin plays a critical role in several cancer models, where it regulates tumor initiation and progression. Recent data from our laboratories have demonstrated that progranulin and its receptor, EphA2, constitute an oncogenic pathway in bladder cancer by promoting motility, invasion and in vivo tumor formation. Progranulin and EphA2 are expressed in mesothelioma cells but their mechanisms of action are not well defined. In addition, there are no data establishing whether the progranulin/EphA2 axis is tumorigenic for mesothelioma cells.

Methods: The expression of progranulin in various mesothelioma cell …

Mesoscale Structure-Function Relationships In Mitochondrial Transcriptional Condensates, Marina Feric, Azadeh Sarfallah, Furqan Dar, Dmitry Temiakov, Rohit V. Pappu, Tom Misteli

Department of Biochemistry and Molecular Biology Faculty Papers

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters …

Pre-Exposure To Mrna-Lnp Inhibits Adaptive Immune Responses And Alters Innate Immune Fitness In An Inheritable Fashion, Zhen Qin, Aurélie Bouteau, Christopher Herbst, Botond Z. Igyártó

Department of Microbiology and Immunology Faculty Papers

Hundreds of millions of SARS-CoV-2 mRNA-LNP vaccine doses have already been administered to humans. However, we lack a comprehensive understanding of the immune effects of this platform. The mRNA-LNP-based SARS-CoV-2 vaccine is highly inflammatory, and its synthetic ionizable lipid component responsible for the induction of inflammation has a long in vivo half-life. Since chronic inflammation can lead to immune exhaustion and non-responsiveness, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness. We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune response, which …

Interplay Between An Atp-Binding Cassette F Protein And The Ribosome From Mycobacterium Tuberculosis, Zhicheng Cui, Xiaojun Li, Joonyoung Shin, Howard Gamper, Ya-Ming Hou, James C Sacchettini, Junjie Zhang

Department of Biochemistry and Molecular Biology Faculty Papers

EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the …

Tera-Seq: True End-To-End Sequencing Of Native Rna Molecules For Transcriptome Characterization, Fadia Ibrahim, Jan Oppelt, Manolis Maragkakis, Zissimos Mourelatos

Department of Biochemistry and Molecular Biology Faculty Papers

Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5′ ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly-and non-polyadenylated RNA molecules and accurately identifies their native 5′ and 3′ ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length …

Structural Basis For +1 Ribosomal Frameshifting During Ef-G-Catalyzed Translocation., Gabriel Demo, Howard Gamper, Anna B. Loveland, Isao Masuda, Christine E. Carbone, Egor Svidritskiy, Ya-Ming Hou, Andrei A. Korostelev

Department of Biochemistry and Molecular Biology Faculty Papers

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near …

Rnase Κ Promotes Robust Pirna Production By Generating 2',3'-Cyclic Phosphate-Containing Precursors, Megumi Shigematsu, Takuya Kawamura, Keisuke Morichika, Natsuko Izumi, Takashi Kiuchi, Shozo Honda, Venetia Pliatsika, Ryuma Matsubara, Isidore Rigoutsos, Susumu Katsuma, Yukihide Tomari, Yohei Kirino

Computational Medicine Center Faculty Papers

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated …

Insights Into Genome Recoding From The Mechanism Of A Classic +1-Frameshifting Trna., Howard Gamper, Haixing Li, Isao Masuda, D. Miklos Robkis, Thomas Christian, Adam B. Conn, Gregor Blaha, E. James Petersson, Ruben L. Gonzalez, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 …

Complete Chemical Structures Of Human Mitochondrial Trnas, Takeo Suzuki, Yuka Yashiro, Ittoku Kikuchi, Yuma Ishigami, Hironori Saito, Ikuya Matsuzawa, Shunpei Okada, Mari Mito, Shintaro Iwasaki, Ding Ma, Xuewei Zhao, Kana Asano, Huan Lin, Yohei Kirino, Yuriko Sakaguchi, Tsutomu Suzuki

Computational Medicine Center Faculty Papers

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 …

Identification Of Early Gene Expression Changes In Primary Cultured Neurons Treated With Topoisomerase I Poisons., Sharyn L. Rossi, Casey J. Lumpkin, Ashlee W. Harris, Jennifer Holbrook, Cinsley Gentillon, Suzanne M. Mccahan, Wenlan Wang, Matthew E.R. Butchbach

Department of Pediatrics Faculty Papers

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and β-lapachone (β-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify …

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.

Differentiation State-Specific Mitochondrial Dynamic Regulatory Networks Are Revealed By Global Transcriptional Analysis Of The Developing Chicken Lens., Daniel Chauss, Subhasree Basu, Suren Rajakaruna, Zhiwei Ma, Victoria Gau, Sara Anastas, Lisa A. Brennan, J. Fielding Hejtmancik, A. Sue Menko, Marc Kantorow

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium …

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 …

Conservation Of Structure And Mechanism By Trm5 Enzymes., Thomas Christian, Howard Gamper, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Enzymes of the Trm5 family catalyze methyl transfer from S-adenosyl methionine (AdoMet) to the N¹ of G37 to synthesize m¹ G37-tRNA as a critical determinant to prevent ribosome frameshift errors. Trm5 is specific to eukaryotes and archaea, and it is unrelated in evolution from the bacterial counterpart TrmD, which is a leading anti-bacterial target. The successful targeting of TrmD requires detailed information on Trm5 to avoid cross-species inhibition. However, most information on Trm5 is derived from studies of the archaeal enzyme Methanococcus jannaschii (MjTrm5), whereas little information is available for eukaryotic enzymes. Here we use human Trm5 (Homo sapiens; HsTrm5) …

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 …

Protein Synthesis Factors (Rf1, Rf2, Rf3, Rrf, And Tmrna) And Peptidyl-Trna Hydrolase Rescue Stalled Ribosomes At Sense Codons., Serafín Vivanco-Domínguez, José Bueno-Martínez, Gloria León-Avila, Nobuhiro Iwakura, Akira Kaji, Hideko Kaji, Gabriel Guarneros

Department of Biochemistry and Molecular Biology Faculty Papers

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of …

Decorin-Mediated Inhibition Of Colorectal Cancer Growth And Migration Is Associated With E-Cadherin In Vitro And In Mice., Xiuli Bi, Nicole M Pohl, Zhibin Qian, George R Yang, Yuan Gou, Grace Guzman, Andre Kajdacsy-Balla, Renato V Iozzo, Wancai Yang

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Previous studies have shown that decorin expression is significantly reduced in colorectal cancer tissues and cancer cells, and genetic deletion of the decorin gene is sufficient to cause intestinal tumor formation in mice, resulting from a downregulation of p21, p27(kip1) and E-cadherin and an upregulation of β-catenin signaling [Bi,X. et al. (2008) Genetic deficiency of decorin causes intestinal tumor formation through disruption of intestinal cell maturation. Carcinogenesis, 29, 1435-1440]. However, the regulation of E-cadherin by decorin and its implication in cancer formation and metastasis is largely unknown. Using a decorin knockout mouse model (Dcn(-/-) mice) and manipulated expression of decorin …

Effect Of Protein Kinase C Delta (Pkc-Δ) Inhibition On The Transcriptome Of Normal And Systemic Sclerosis Human Dermal Fibroblasts In Vitro., Peter J Wermuth, Sankar Addya, Sergio A Jimenez

Jefferson Institute of Molecular Medicine Papers and Presentations

Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 …

Abca12-Mediated Lipid Transport And Snap29-Dependent Trafficking Of Lamellar Granules Are Crucial For Epidermal Morphogenesis In A Zebrafish Model Of Ichthyosis., Qiaoli Li, Michael Frank, Masashi Akiyama, Shiu-Ying Ho, Hiroshi Shimizu, Christine Thisse, Bernard Thisse, Eli Sprecher, Jouni Uitto

Department of Dermatology and Cutaneous Biology Faculty Papers

Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5-6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and …

The Interplay Between Nf-Kappab And E2f1 Coordinately Regulates Inflammation And Metabolism In Human Cardiac Cells., Xavier Palomer, David Álvarez-Guardia, Mercy M Davidson, Tung O Chan, Arthur M Feldman, Manuel Vázquez-Carrera

Department of Medicine Faculty Papers

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, …

Mechanism Of N-Methylation By The Trna M1g37 Methyltransferase Trm5., Thomas Christian, Georges Lahoud, Cuiping Liu, Katherine Hoffmann, John J Perona, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one …

Pp32 (Anp32a) Expression Inhibits Pancreatic Cancer Cell Growth And Induces Gemcitabine Resistance By Disrupting Hur Binding To Mrnas., Timothy K Williams, Christina L Costantino, Nikolai A Bildzukewicz, Nathan G Richards, David W Rittenhouse, Lisa Einstein, Joseph A Cozzitorto, Judith C Keen, Abhijit Dasgupta, Myriam Gorospe, Gregory E Gonye, Charles J Yeo, Agnieszka K Witkiewicz, Jonathan R Brody

Department of Surgery Faculty Papers

The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, …

Control Of Catalytic Cycle By A Pair Of Analogous Trna Modification Enzymes., Thomas Christian, Georges Lahoud, Cuiping Liu, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here, we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at …

Control Of Catalytic Cycle By A Pair Of Analogous Trna Modification Enzymes., Thomas Christian, Georges Lahoud, Cuiping Liu, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here, we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at …

Ribosome Recycling Step In Yeast Cytoplasmic Protein Synthesis Is Catalyzed By Eef3 And Atp., Shinya Kurata, Klaus H Nielsen, Sarah F Mitchell, Jon R Lorsch, Akira Kaji, Hideko Kaji

Department of Biochemistry and Molecular Biology Faculty Papers

After each round of protein biosynthesis, the posttermination complex (PoTC) consisting of a ribosome, mRNA, and tRNA must be disassembled into its components for a new round of translation. Here, we show that a Saccharomyces cerevisiae model PoTC was disassembled by ATP and eukaryotic elongation factor 3 (eEF3). GTP or ITP functioned with less efficiency and adenosine 5gamma'-(beta,gamma-imido)triphosphate did not function at all. The k(cat) of eEF3 was 1.12 min(-1), which is comparable to that of the in vitro initiation step. The disassembly reaction was inhibited by aminoglycosides and cycloheximide. The subunits formed from the yeast model PoTC remained separated …