Life Sciences Commons^™

Transcriptional Regulation Factors Of The Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (Slc25a13): Usf1 As Basal Factor And Foxa2 As Activator In Liver Cells, Paolo Convertini, Simona Todisco, Francesco De Santis, Ilaria Pappalardo, Dominga Iacobazzi, Maria Antonietta Castiglione Morelli, Yvonne N. Fondufe-Mittendorf, Giuseppe Martelli, Ferdinando Palmieri, Vittoria Infantino

Molecular and Cellular Biochemistry Faculty Publications

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (−442 bp/−19 bp), as well as an enhancer region (−968 bp/−768 bp). Furthermore, with different …

Stress-Induced Epinephrine Enhances Lactate Dehydrogenase A And Promotes Breast Cancer Stem-Like Cells, Bai Cui, Yuanyuan Luo, Pengfei Tian, Fei Peng, Jinxin Lu, Yongliang Yang, Qitong Su, Bing Liu, Jiachuan Yu, Xi Luo, Liu Yin, Wei Cheng, Fan An, Bin He, Dapeng Liang, Sijin Wu, Peng Chu, Luyao Song, Xinyu Liu, Huandong Luo, Binhua P. Zhou

Molecular and Cellular Biochemistry Faculty Publications

Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress–induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A–dependent (LDHA-dependent) metabolic rewiring. Chronic stress–induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress–induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological …

Immunization Of Alpacas (Lama Pacos) With Protein Antigens And Production Of Antigen-Specific Single Domain Antibodies, K. Martin Chow, Sidney W. Whiteheart, Jeffrey R. Smiley, Savita Sharma, Kathy Boaz, Meggie J. Coleman, Alvina Maynard, Louis B. Hersh, Craig W. Vander Kooi

Molecular and Cellular Biochemistry Faculty Publications

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies …

Targeting The Brd4/Foxo3a/Cdk6 Axis Sensitizes Akt Inhibition In Luminal Breast Cancer, Jingyi Liu, Weijie Guo, Zhibing Duan, Lei Zeng, Yadi Wu, Yule Chen, Fang Tai, Yifan Wang, Yiwei Lin, Qiang Zhang, Yanling He, Jiong Deng, Rachel L. Stewart, Chi Wang, Pengnian Charles Lin, Saghi Ghaffari, B. Mark Evers, Suling Liu, Ming-Ming Zhou, Binhua P. Zhou, Jian Shi

Molecular and Cellular Biochemistry Faculty Publications

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or …

Structure Of The Mouse Trpc4 Ion Channel, Jingjing Duan, Jian Li, Bo Zeng, Gui-Lan Chen, Xiaogang Peng, Yixing Zhang, Jianbin Wang, David E. Clapham, Zongli Li, Jin Zhang

Molecular and Cellular Biochemistry Faculty Publications

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP …

Structure Of Full-Length Human Trpm4, Jingjing Duan, Zongli Li, Jian Li, Ana Santa-Cruz, Silvia Sanchez-Martinez, Jin Zhang, David E. Clapham

Molecular and Cellular Biochemistry Faculty Publications

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na⁺-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist …

Kruppel-Like Factor 4-Dependent Staufen1-Mediated Mrna Decay Regulates Cortical Neurogenesis, Byoung-San Moon, Jinlun Bai, Mingyang Cai, Chunming Liu, Jiandang Shi, Wange Lu

Molecular and Cellular Biochemistry Faculty Publications

Kruppel-like factor 4 (Klf4) is a zinc-finger-containing protein that plays a critical role in diverse cellular physiology. While most of these functions attribute to its role as a transcription factor, it is postulated that Klf4 may play a role other than transcriptional regulation. Here we demonstrate that Klf4 loss in neural progenitor cells (NPCs) leads to increased neurogenesis and reduced self-renewal in mice. In addition, Klf4 interacts with RNA-binding protein Staufen1 (Stau1) and RNA helicase Ddx5/17. They function together as a complex to maintain NPC self-renewal. We report that Klf4 promotes Stau1 recruitment to the 3′-untranslated region of neurogenesis-associated mRNAs, …

Selective Inhibition Of Ctcf Binding By Ias Directs Tet-Mediated Reprogramming Of 5-Hydroxymethylation Patterns In Ias-Transformed Cells, Matthew Rea, Tyler Gripshover, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Methylation at cytosine (5mC) is a fundamental epigenetic DNA modification recently associated with iAs-mediated carcinogenesis. In contrast, the role of 5-hydroxymethylcytosine (5hmC), the oxidation product of 5mC in iAs-mediated carcinogenesis is unknown. Here we assess the hydroxymethylome in iAs-transformed cells, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks. Moreover, this pathologic iAs-mediated carcinogenesis is characterized by a shift toward a higher hydroxymethylation pattern genome-wide. At specific promoters, hydroxymethylation correlated with increased gene expression. Furthermore, this increase in hydroxymethylation occurs concurrently with an upregulation of ten-eleven translocation (TET) enzymes that oxidize 5-methylcytosine (5mC) in DNA. To …

Human Metapneumovirus Induces Formation Of Inclusion Bodies For Efficient Genome Replication And Transcription, Nicolás P. Cifuentes-Muñoz, Jean Branttie, Kerri Beth Slaughter, Rebecca Ellis Dutch

Molecular and Cellular Biochemistry Faculty Publications

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA …

Tox Regulates Growth, Dna Repair, And Genomic Instability In T-Cell Acute Lymphoblastic Leukemia, Riadh Lobbardi, Jordan Pinder, Barbara Martinez-Pastor, Marina Theodorou, Jessica S. Blackburn, Brian J. Abraham, Yuka Namiki, Marc Mansour, Nouran S. Abdelfattah, Aleksey Molodtsov, Gabriela Alexe, Debra Toiber, Manon De Waard, Esha Jain, Myriam Boukhali, Mattia Lion, Deepak Bhere, Khalid Shah, Alejandro Gutierrez, Kimberly Stegmaier, Lewis B. Silverman, Ruslan I. Sadreyev, John M. Asara, Marjorie A. Oettinger, Wilhelm Haas, A. Thomas Look, Richard A. Young, Raul Mostoslavsky, Graham Dellaire, David M. Langenau

Molecular and Cellular Biochemistry Faculty Publications

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection–associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. …

Nanoparticle Delivery Of Mir-34a Eradicates Long-Term-Cultured Breast Cancer Stem Cells Via Targeting C22orf28 Directly, Xiaoti Lin, Weiyu Chen, Fengqin Wei, Binhua P. Zhou, Mien-Chie Hung, Xiaoming Xie

Molecular and Cellular Biochemistry Faculty Publications

Rationale: Cancer stem cells (CSCs) have been implicated as the seeds of therapeutic resistance and metastasis, due to their unique abilities of self-renew, wide differentiation potentials and resistance to most conventional therapies. It is a proactive strategy for cancer therapy to eradicate CSCs. Methods: Tumor tissue-derived breast CSCs (BCSC), including XM322 and XM607, were isolated by fluorescence-activated cell sorting (FACS); while cell line-derived BCSC, including MDA-MB-231.SC and MCF-7.SC, were purified by magnetic-activated cell sorting (MACS). Analyses of microRNA and mRNA expression array profiles were performed in multiple breast cell lines. The mentioned nanoparticles were constructed following the standard molecular cloning …

Transient And Permanent Changes In Dna Methylation Patterns In Inorganic Arsenic-Mediated Epithelial-To-Mesenchymal Transition, Meredith Eckstein, Matthew Rea, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells …

The Activity Of The Serotonin Receptor 2c Is Regulated By Alternative Splicing, Stefan Stamm, Samuel B. Gruber, Alexander G. Rabchevsky, Ronald B. Emeson

Molecular and Cellular Biochemistry Faculty Publications

The central nervous system-specific serotonin receptor 2C (5HT2C) controls key physiological functions, such as food intake, anxiety, and motoneuron activity. Its deregulation is involved in depression, suicidal behavior, and spasticity, making it the target for antipsychotic drugs, appetite controlling substances, and possibly anti-spasm agents. Through alternative pre-mRNA splicing and RNA editing, the 5HT2C gene generates at least 33 mRNA isoforms encoding 25 proteins. The 5HT2C is a G-protein coupled receptor that signals through phospholipase C, influencing the expression of immediate/early genes like c-fos. Most 5HT2C isoforms show constitutive activity, i.e., signal without ligand binding. The constitutive activity of 5HT2C is …

Mutations In The Transmembrane Domain And Cytoplasmic Tail Of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly, Nicolás P. Cifuentes-Muñoz, Weina Sun, Greeshma Ray, Phuong Tieu Schmitt, Stacy Webb, Kathleen Gibson, Rebecca Ellis Dutch, Anthony P. Schmitt

Molecular and Cellular Biochemistry Faculty Publications

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking …

C/D-Box Snornas Form Methylating And Non-Methylating Ribonucleoprotein Complexes: Old Dogs Show New Tricks, Marina Falaleeva, Justin R. Welden, Marilyn J. Duncan, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

C/D box snoRNAs (SNORDs) are an abundantly expressed class of short, non‐coding RNAs that have been long known to perform 2′‐O‐methylation of rRNAs. However, approximately half of human SNORDs have no predictable rRNA targets, and numerous SNORDs have been associated with diseases that show no defects in rRNAs, among them Prader‐Willi syndrome, Duplication 15q syndrome and cancer. This apparent discrepancy has been addressed by recent studies showing that SNORDs can act to regulate pre‐mRNA alternative splicing, mRNA abundance, activate enzymes, and be processed into shorter ncRNAs resembling miRNAs and piRNAs. Furthermore, recent biochemical studies have shown that a given SNORD …

Perspectives And Expectations In Structural Bioinformatics Of Metalloproteins, Sen Yao, Robert M. Flight, Eric C. Rouchka, Hunter N. B. Moseley

Molecular and Cellular Biochemistry Faculty Publications

Recent papers highlight the presence of large numbers of compressed angles in metal ion coordination geometries for metalloprotein entries in the worldwide Protein Data Bank, due mainly to multidentate coordination. The prevalence of these compressed angles has raised the controversial idea that significantly populated aberrant or even novel coordination geometries may exist. Some of these papers have undergone severe criticism, apparently due to views held that only canonical coordination geometries exist in significant numbers. While criticism of controversial ideas is warranted and to be expected, we believe that a line was crossed where unfair criticism was put forth to discredit …

Editorial: Platelet Secretion, Brian Storrie, Sidney W. Whiteheart

Molecular and Cellular Biochemistry Faculty Publications

No abstract provided.

The Nuts And Bolts Of The Platelet Release Reaction, Smita Joshi, Sidney W. Whiteheart

Molecular and Cellular Biochemistry Faculty Publications

Secretion is essential to many of the roles that platelets play in the vasculature, e.g., thrombosis, angiogenesis, and inflammation, enabling platelets to modulate the microenvironment at sites of vascular lesions with a myriad of bioactive molecules stored in their granules. Past studies demonstrate that granule cargo release is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins, which are required for granule-plasma membrane fusion. Several SNARE regulators, which control when, where, and how the SNAREs interact, have been identified in platelets. Additionally, platelet SNAREs are controlled by post-translational modifications, e.g., phosphorylation and acylation. Although there have been many …

Lipophosphoglycans From Leishmania Amazonensis Strains Display Immunomodulatory Properties Via Tlr4 And Do Not Affect Sand Fly Infection, Paula M. Nogueira, Rafael R. Assis, Ana C. Torrecilhas, Elvira M. Saraiva, Natália L. Pessoa, Marco A. Campos, Eric F. Marialva, Cláudia M. Ríos-Velasquez, Felipe A. Pessoa, Nágila F. Secundino, Jerônimo N. Rugani, Elsa Nieves, Salvatore J. Turco, Maria N. Melo, Rodrigo P. Soares

Molecular and Cellular Biochemistry Faculty Publications

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of …

Quantitative Mass Spectrometry Reveals Changes In Histone H2b Variants As Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation, Matthew Rea, Tingting Jiang, Rebekah Eleazer, Meredith Eckstein, Alan G. Marshall, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here …

Twist-Mediated Epithelial-Mesenchymal Transition Promotes Breast Tumor Cell Invasion Via Inhibition Of Hippo Pathway, Yifan Wang, Jingyi Liu, Xuhua Ying, Pengnian Charles Lin, Binhua P. Zhou

Molecular and Cellular Biochemistry Faculty Publications

Twist is a key transcription factor for Epithelial-mesenchymal transition (EMT), which is a cellular de-differentiation program that promotes invasion and metastasis, confers tumor cells with cancer stem cell (CSC)-like characteristics, and increases therapeutic resistance. However, the mechanisms that facilitate the functions of Twist remain unclear. Here we report that Twist overexpression increased expression of PAR1, an upstream regulator of the Hippo pathway; PAR1 promotes invasion, migration, and CSC-like properties in breast cancer by activating the transcriptional co-activator TAZ. Our study indicates that Hippo pathway inhibition is required for the increased migratory and invasiveness ability of breast cancer cells in Twist-mediated …

Arf6 Controls Platelet Spreading And Clot Retraction Via Integrin ΑIibΒ3 Trafficking, Yunjie Huang, Smita Joshi, Binggang Xiang, Yasunori Kanaho, Zhenyu Li, Beth A. Bouchard, Carole L. Moncman, Sidney W. Whiteheart

Molecular and Cellular Biochemistry Faculty Publications

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects α_IIbβ₃-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets …

P-Rex1 Promotes Resistance To Vegf/Vegfr-Targeted Therapy In Prostate Cancer, Hira Lal Goel, Bryan Pursell, Leonard D. Shultz, Dale L. Greiner, Rolf A Brekken, Craig W. Vander Kooi, Arthur M. Mercurio

Molecular and Cellular Biochemistry Faculty Publications

Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN^pc−/− transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the …

Imaging Tumour Cell Heterogeneity Following Cell Transplantation Into Optically Clear Immune-Deficient Zebrafish, Qin Tang, John C. Moore, Myron S. Ignatius, Inês M. Tenente, Madeline N. Hayes, Elaine G. Garcia, Nora Torres Yordán, Caitlin Bourque, Shuning He, Jessica S. Blackburn, A. Thomas Look, Yariv Houvras, David M. Langenau

Molecular and Cellular Biochemistry Faculty Publications

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2^E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal …

Linker Histone H1 And H3k56 Acetylation Are Antagonistic Regulators Of Nucleosome Dynamics, Morgan Bernier, Yi Luo, Kingsley C. Nwokelo, Michelle Goodwin, Sarah J. Dreher, Pei Zhang, Mark R. Parthun, Yvonne N. Fondufe-Mittendorf, Jennifer J. Ottesen, Michael G. Poirier

Molecular and Cellular Biochemistry Faculty Publications

H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF …

Differential Impact Of Lpg-And Pg-Deficient Leishmania Major Mutants On The Immune Response Of Human Dendritic Cells, Michelle A. Favila, Nicholas S. Geraci, Asha Jayakumar, Suzanne Hickerson, Janet Mostrom, Salvatore J. Turco, Stephen M. Beverley, Mary Ann Mcdowell

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.

METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective …

Mechanistic Insights Into Glucan Phosphatase Activity Against Polyglucan Substrates, David A. Meekins, Madushi Raththagala, Kyle D. Auger, Benjamin D. Turner, Diana Santelia, Oliver Kötting, Matthew S. Gentry, Craig W. Vander Kooi

Molecular and Cellular Biochemistry Faculty Publications

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. …

Autophagy Is Induced Upon Platelet Activation And Is Essential For Hemostasis And Thrombosis, Madhu M. Ouseph, Yunjie Huang, Meenakshi Banerjee, Smita Joshi, Laura Macdonald, Yu Zhong, Huijuan Liu, Xianting Li, Binggang Xiang, Guoying Zhang, Masaaki Komatsu, Zhenyu Yue, Zhenyu Li, Brian Storrie, Sidney W. Whiteheart, Qing Jun Wang

Molecular and Cellular Biochemistry Faculty Publications

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling …

Genome-Wide Profiling Of Parp1 Reveals An Interplay With Gene Regulatory Regions And Dna Methylation, Narasimharao Nalabothula, Taha Al-Jumaily, Abdallah M. Eteleeb, Robert M. Flight, Shao Xiaorong, Hunter Moseley, Eric C. Rouchka, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone …

Intra-Domain Cross-Talk Regulates Serine-Arginine Protein Kinase 1-Dependent Phosphorylation And Splicing Function Of Transformer 2Β1, Michael A. Jamros, Brandon E. Aubol, Malik M. Keshwani, Zhaiyi Zhang, Stefan Stamm, Joseph A. Adams

Molecular and Cellular Biochemistry Faculty Publications

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated …