Medicine and Health Sciences Commons^™

Zur And Zinc Increase Expression Of E. Coli Ribosomal Protein L31 Through Rna-Mediated Repression Of The Repressor L31p, Rebecca A Rasmussen, Suning Wang, Jeannie M Camarillo, Victoria Sosnowski, Byoung-Kyu Cho, Young Ah Goo, Julius B Lucks, Thomas V O'Halloran

2020-Current year OA Pubs

Bacteria can adapt in response to numerous stress conditions. One such stress condition is zinc depletion. The zinc-sensing transcription factor Zur regulates the way numerous bacterial species respond to severe changes in zinc availability. Under zinc sufficient conditions, Zn-loaded Zur (Zn2-Zur) is well-known to repress transcription of genes encoding zinc uptake transporters and paralogues of a few ribosomal proteins. Here, we report the discovery and mechanistic basis for the ability of Zur to up-regulate expression of the ribosomal protein L31 in response to zinc in E. coli. Through genetic mutations and reporter gene assays, we find that Zur achieves the …

Prodrugs Of A 1-Hydroxy-2-Oxopiperidin-3-Yl Phosphonate Enolase Inhibitor For The Treatment Of Eno1-Deleted Cancers, Victoria C Yan, Cong-Dat Pham, Elliot S Ballato, Kristine L Yang, Kenisha Arthur, Sunada Khadka, Yasaman Barekatain, Prakriti Shrestha, Theresa Tran, Anton H Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J Ackroyd, Dimitra K Georgiou, Steven W Millward, Florian L Muller

Journal Articles

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma …

Regulation Of Human Cortical Interneuron Development By The Chromatin Remodeling Protein Chd2, E M A Lewis, G Chapman, K Kaushik, J Determan, I Antony, K Meganathan, M Narasimhan, P Gontarz, B Zhang, K L Kroll

2020-Current year OA Pubs

Mutations in the chromodomain helicase DNA binding protein 2 (CHD2) gene are associated with neurodevelopmental disorders. However, mechanisms by which CHD2 regulates human brain development remain largely uncharacterized. Here, we used a human embryonic stem cell model of cortical interneuron (hcIN) development to elucidate its roles in this process. We identified genome-wide CHD2 binding profiles during hcIN differentiation, defining direct CHD2 targets related to neurogenesis in hcIN progenitors and to neuronal function in hcINs. CHD2 bound sites were frequently coenriched with histone H3 lysine 27 acetylation (H3K27ac) and associated with high gene expression, indicating roles for CHD2 in promoting gene …

Smyd3 Impedes Small Cell Lung Cancer Sensitivity To Alkylation Damage Through Rnf113a Methylation-Phosphorylation Cross-Talk, Valentina Lukinović, Clement Oyeniran, Ning Tsao, Joshua R Brickner, Rebecca Rodell, Nima Mosammaparast, Et Al

2020-Current year OA Pubs

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of …

Recognition Of The Tdp-43 Nuclear Localization Signal By Importin Α1/Β, Steven G Doll, Hamed Meshkin, Alexander J Bryer, Fenglin Li, Ying-Hui Ko, Ravi K Lokareddy, Richard E Gillilan, Kushol Gupta, Juan R Perilla, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we study how importin α1/β recognizes TDP-43 bipartite nuclear localization signal (NLS). We find that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. NLS binding results in steric clashes with the C terminus of importin α1 that disrupts the TDP-43 N-terminal domain (NTD) dimerization interface. A putative phosphorylation site in the proximity of TDP-43 R83 at the minor NLS site destabilizes binding to importins by reducing …

Loss Of Stathmin-2, A Hallmark Of Tdp-43-Associated Als, Causes Motor Neuropathy, Kelsey L Krus, Amy Strickland, Yurie Yamada, Laura Devault, Robert E Schmidt, A Joseph Bloom, Jeffrey Milbrandt, Aaron Diantonio

2020-Current year OA Pubs

TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most patients with amyotrophic lateral sclerosis (ALS). To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 knockout heterozygous mice, which better model the partial loss …

A Genome-Wide Crispr-Cas9 Knockout Screen Identifies Essential And Growth-Restricting Genes In Human Trophoblast Stem Cells, Chen Dong, Shuhua Fu, Rowan M Karvas, Brian Chew, Laura A Fischer, Xiaoyun Xing, Jessica K Harrison, Pooja Popli, Ramakrishna Kommagani, Ting Wang, Bo Zhang, Thorold W Theunissen

2020-Current year OA Pubs

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, …

Directed Mutational Scanning Reveals A Balance Between Acidic And Hydrophobic Residues In Strong Human Activation Domains, Max V Staller, Eddie Ramirez, Sanjana R Kotha, Alex S Holehouse, Rohit V Pappu, Barak A Cohen

2020-Current year OA Pubs

Acidic activation domains are intrinsically disordered regions of the transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features that control activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a high-throughput assay in human cell culture. We found that strong activation domain activity requires a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of acidic activation domains that scans the human proteome for clusters of …

Vcp Suppresses Proteopathic Seeding In Neurons, Jiang Zhu, Sara Pittman, Dhruva Dhavale, Jessica N Patterson, Mohamed Salman Kaleelurrrahuman, William J Buscher, Paul Kotzbauer, Albert A Davis, Conrad Weihl, Et Al.

2020-Current year OA Pubs

BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43.

METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary …

Mechanisms Of Mitochondrial Promoter Recognition In Humans And Other Mammalian Species, Angelica Zamudio-Ochoa, Yaroslav I Morozov, Azadeh Sarfallah, Michael Anikin, Dmitry Temiakov

Department of Biochemistry and Molecular Biology Faculty Papers

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of …

Nanorings To Probe Mechanical Stress Of Single-Stranded Dna Mediated By The Dna Duplex, Karen Zagorski, Tommy Stormberg, Mohtadin Hashemi, Anatoly B. Kolomeisky, Yuri L. Lyubchenko

Journal Articles: Pharmaceutical Sciences

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic …