Biochemistry, Biophysics, and Structural Biology Commons^™

Lesion-Specific Dna-Binding And Repair Activities Of Human O⁶-Alkylguanine Dna Alkyltransferase, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Binding experiments with alkyl-transfer-active and -inactive mutants of human O⁶-alkylguanine DNA alkyltransferase (AGT) show that it forms an O⁶-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with …

C6 Pyridinium Ceramide Influences Alternative Pre-Mrna Splicing By Inhibiting Protein Phosphatase-1, Chiranthani Sumanasekera, Olga Kelemen, Monique Beullens, Brandon E. Aubol, Joseph A. Adams, Manjula Sunkara, Andrew J. Morris, Mathieu Bollen, Athena Andreadis, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic …

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Dartmouth Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs …

Planning Combinatorial Disulfide Cross-Links For Protein Fold Determination, Fei Xiong, Alan M Friedman, Chris Bailey-Kellogg

Dartmouth Scholarship

Fold recognition techniques take advantage of the limited number of overall structural organizations, and have become increasingly effective at identifying the fold of a given target sequence. However, in the absence of sufficient sequence identity, it remains difficult for fold recognition methods to always select the correct model. While a native-like model is often among a pool of highly ranked models, it is not necessarily the highest-ranked one, and the model rankings depend sensitively on the scoring function used. Structure elucidation methods can then be employed to decide among the models based on relatively rapid biochemical/biophysical experiments.

Differential Il-21 Signaling In Apcs Leads To Disparate Th17 Differentiation In Diabetes-Susceptible Nod And Diabetes-Resistant Nod.Idd3 Mice., Sue M. Liu, David H. Lee, Jenna M. Sullivan, Denise Chung, Anneli Jäger, Bennett O V. Shum, Nora E. Sarvetnick, Ana C. Anderson, Vijay K. Kuchroo

Journal Articles: Regenerative Medicine

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with …

Splice Variant–Specific Cellular Function Of The Formin Inf2 In Maintenance Of Golgi Architecture, Vinay Ramabhadran, Farida Korobova, Gilbert J. Rahme, Henry N. Higgs

Dartmouth Scholarship

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized …

Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs

Dartmouth Scholarship

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Stomatin-Like Protein 2 Binds Cardiolipin And Regulates Mitochondrial Biogenesis And Function., Darah Christie, Caitlin D Lemke, Isaac M Elias, Luan A Chau, Mark G Kirchhof, Bo Li, Eric H Ball, Stanley D Dunn, Grant M Hatch, Joaquín Madrenas

Biochemistry Publications

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched …

Laforin, A Dual Specificity Phosphatase Involved In Lafora Disease, Is Present Mainly As Monomeric Form With Full Phosphatase Activity, Vikas V. Dukhande, Devin M. Rogers, Carlos Romá-Mateo, Jordi Donderis, Alberto Marina, Adam O. Taylor, Pascual Sanz, Matthew S. Gentry

Molecular and Cellular Biochemistry Faculty Publications

Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, …

Serum- And Glucocorticoid-Induced Kinase 3 In Recycling Endosomes Mediates Acute Activation Of Na+/H+ Exchanger Nhe3 By Glucocorticoids, Peijian He, Sei-Jung Lee, Songbai Lin, Ursula Seidler, Florian Lang, Geza Fejes-Toth, Aniko Naray-Fejes-Toth, C. Chris Yun

Dartmouth Scholarship

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or …

Lafora Disease E3-Ubiquitin Ligase Malin Is Related To Trim32 At Both The Phylogenetic And Functional Level, Carlos Romá-Mateo, Daniel Moreno, Santiago Vernia, Teresa Rubio, Travis M. Bridges, Matthew S. Gentry, Pascual Sanz

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin.

RESULTS: After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming …

Digeorge Critical Region 8 (Dgcr8) Is A Double-Cysteine-Ligated Heme Protein., Ian Barr, Aaron T. Smith, Rachel Senturia, Yanqiu Chen, Brooke D. Scheidemantle, Judith N. Burstyn, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 …

Nogo-B Receptor Is Necessary For Cellular Dolichol Biosynthesis And Protein N-Glycosylation., Kenneth D Harrison, Eon Joo Park, Ningguo Gao, Andrew Kuo, Jeffrey S. Rush, Charles J Waechter, Mark A Lehrman, William C Sessa

Molecular and Cellular Biochemistry Faculty Publications

Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of …

Decreased Stability And Increased Formation Of Soluble Aggregates By Immature Superoxide Dismutase Do Not Account For Disease Severity In Als., Kenrick A Vassall, Helen R Stubbs, Heather A Primmer, Ming Sze Tong, Sarah M Sullivan, Ryan Sobering, Saipraveen Srinivasan, Lee-Ann K Briere, Stanley D Dunn, Wilfredo Colón, Elizabeth M Meiering

Biochemistry Publications

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic …

Activation Of Β-Catenin And Akt Pathways By Twist Are Critical For The Maintenance Of Emt Associated Cancer Stem Cell-Like Characters, Junlin Li, Binhua P. Zhou

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Epithelial-mesenchymal transition (EMT) not only confers tumor cells with a distinct advantage for metastatic dissemination, but also it provides those cells with cancer stem cell-like characters for proliferation and drug resistance. However, the molecular mechanism for maintenance of these stem cell-like traits remains unclear.

METHODS: In this study, we induced EMT in breast cancer MCF7 and cervical cancer Hela cells with expression of Twist, a key transcriptional factor of EMT. The morphological changes associated with EMT were analyzed by immunofluorescent staining and Western blotting. The stem cell-like traits associated with EMT were determined by tumorsphere-formation and expression of ALDH1 …

Requirement For Golgi-Localized Pi(4)P In Fusion Of Copii Vesicles With Golgi Compartments, Andres Lorente-Rodriguez, Charles Barlowe

Dartmouth Scholarship

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) …

The Mir-15/107 Group Of Microrna Genes: Evolutionary Biology, Cellular Functions, And Roles In Human Diseases, John R. Finnerty, Wang-Xia Wang, Sébastien S. Hébert, Bernard R. Wilfred, Guogen Mao, Peter T. Nelson

Pathology and Laboratory Medicine Faculty Publications

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played …

Mechanisms Of Oxidant Generation By Catalase, Diane E. Heck, Michael Shakarjian, Hong-Duck Kim, Jeffrey Laskin, Anna M. Vetrano

NYMC Faculty Publications

The enzyme catalase converts solar radiation into reactive oxidant species (ROS). In this study, we report that several bacterial catalases (hydroperoxidases, HP), including Escherichia coli HP-I and HP-II also generate reactive oxidants in response to ultraviolet B light (UVB). HP-I and HP-II are identical except for the presence of NADPH. We found that only one of the catalases, HPI, produces oxidants in response to UVB light, indicating a potential role for the nucleotide in ROS production. This prompts us to speculate that NADPH may act as a cofactor regulating ROS generation by mammalian catalases. Structural analysis of the NADPH domains …

The Marine Sponge Metabolite Mycothiazole: A Novel Prototype Mitochondrial Complex I Inhibitor., J Brian Morgan, Fakhri Mahdi, Yang Liu, Veena Coothankandaswamy, Mika B. Jekabsons, Tyler A. Johnson, Koneni V. Sashidhara, Phillip Crews, Dale G. Nagle, Yu-Dong Zhou

Natural Sciences and Mathematics | Faculty Scholarship

A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC(50) 1nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP(+), annonaceous acetogenins, piericidin A, and other complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound …

Entry And Fusion Of Emerging Paramyxoviruses, Rebecca Ellis Dutch

Molecular and Cellular Biochemistry Faculty Publications

Paramyxoviruses are a family of non-segmented RNA viruses that includes major human pathogens such as measles virus and respiratory syncytial virus (RSV) and significant animal viruses like rinderpest. In recent years, several new paramyxoviruses have been identified, further increasing the breadth and importance of this viral family. While many elements of the fusion and entry mechanisms of these recently identified pathogens are conserved, there are interesting differences, including variations in receptor binding, cell tropism, fusion (F) protein proteolytic activation, and triggering of membrane fusion. Thus, study of their entry mechanisms has highlighted the diversity of these critical events in the …

Mimosa: A System For Minimotif Annotation, Jay Vyas, Ronald J. Nowling, Thomas Meusburger, David P. Sargeant, Krishna Kadaveru, Michael R. Gryk, Vamsi Kundeti, Sanguthevar Rajasekaran, Martin Schiller

Life Sciences Faculty Research

BACKGROUND:

Minimotifs are short peptide sequences within one protein, which are recognized by other proteins or molecules. While there are now several minimotif databases, they are incomplete. There are reports of many minimotifs in the primary literature, which have yet to be annotated, while entirely novel minimotifs continue to be published on a weekly basis. Our recently proposed function and sequence syntax for minimotifs enables us to build a general tool that will facilitate structured annotation and management of minimotif data from the biomedical literature.

RESULTS:

We have built the MimoSA application for minimotif annotation. The application supports management of …

Partitioning Of Minimotifs Based On Function With Improved Prediction Accuracy, Sanguthevar Rajasekaran, Tian Mi, Jerlin Camilus Merlin, Aaron Oommen, Patrick R. Gradie, Martin R. Schiller

Life Sciences Faculty Research

Background

Minimotifs are short contiguous peptide sequences in proteins that are known to have a function in at least one other protein. One of the principal limitations in minimotif prediction is that false positives limit the usefulness of this approach. As a step toward resolving this problem we have built, implemented, and tested a new data-driven algorithm that reduces false-positive predictions.

Methodology/Principal Findings

Certain domains and minimotifs are known to be strongly associated with a known cellular process or molecular function. Therefore, we hypothesized that by restricting minimotif predictions to those where the minimotif containing protein and target protein have …

Venn, A Tool For Titrating Sequence Conservation Onto Protein Structures, Jay Vyas, Michael R. Gryk, Martin R. Schiller

Life Sciences Faculty Research

Residue conservation is an important, established method for inferring protein function, modularity and specificity. It is important to recognize that it is the 3D spatial orientation of residues that drives sequence conservation. Considering this, we have built a new computational tool, VENN that allows researchers to interactively and graphically titrate sequence homology onto surface representations of protein structures. Our proposed titration strategies reveal critical details that are not readily identified using other existing tools. Analyses of a bZIP transcription factor and receptor recognition of Fibroblast Growth Factor using VENN revealed key specificity determinants. Weblink: http://sbtools.uchc.edu/venn/.

Conservation Of The Glucan Phosphatase Laforin Is Linked To Rates Of Molecular Evolution And The Glucan Metabolism Of The Organism, Matthew S. Gentry, Rachel M. Pace

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for approximately 50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.

RESULTS: Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate …

Human Cerebral Neuropathology Of Type 2 Diabetes Mellitus, Peter T. Nelson, Charles D. Smith, Erin L. Abner, Frederick A. Schmitt, Stephen W. Scheff, Gregory J. Davis, Jeffrey N. Keller, Gregory A. Jicha, Daron Davis, Wang-Xia Wang, Adria Hartman, Douglas G. Katz, William R. Markesbery

Pathology and Laboratory Medicine Faculty Publications

The cerebral neuropathology of Type 2 diabetes (CNDM2) has not been positively defined. This review includes a description of CNDM2 research from before the ‘Pubmed Era’. Recent neuroimaging studies have focused on cerebrovascular and white matter pathology. These and prior studies about cerebrovascular histopathology in diabetes are reviewed. Evidence is also described for and against the link between CNDM2 and Alzheimer's disease pathogenesis. To study this matter directly, we evaluated data from University of Kentucky Alzheimer's Disease Center (UK ADC) patients recruited while non-demented and followed longitudinally. Of patients who had come to autopsy (N = 234), 139 met …

Polyglutamine Disruption Of The Huntingtin Exon 1 N Terminus Triggers A Complex Aggregation Mechanism, Ashwani K. Thakur, Murali Jayaraman, Rakesh Mishra, Monika Thakur, Veronique M. Chellgren, In-Ja L Byeon, Dalaver H. Anjum, Ravindra Kodali, Trevor P. Creamer, James F. Conway, Angela M. Gronenborn, Ronald Wetzel

Molecular and Cellular Biochemistry Faculty Publications

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT^NT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT^NT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT^NT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT^NT cores and exposed polyQ. In a second …

Sumoylation Regulates Lamin A Function And Is Lost In Lamin A Mutants Associated With Familial Cardiomyopathies, Yu-Qian Zhang, Kevin D. Sarge

Molecular and Cellular Biochemistry Faculty Publications

Lamin A mutations cause many diseases, including cardiomyopathies and Progeria Syndrome. The covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides regulates the function of many proteins. Until now, no examples of human disease-causing mutations that occur within a sumoylation consensus sequence and alter sumoylation were known. We show that lamin A is sumoylated at lysine 201 and that two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. E203 occupies the conserved +2 position in the sumoylation consensus Psi KXE. Lamin A mutants E203G, E203K, and K201R all exhibit a similar aberrant subcellular localization and …

Examining The Link Between Chromosomal Instability And Aneuploidy In Human Cells, Sarah L. Thompson, Duane A. Compton

Dartmouth Scholarship

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule–chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, …

Il-21 Limits Peripheral Lymphocyte Numbers Through T Cell Homeostatic Mechanisms., Shrimati Datta, Nora E. Sarvetnick

Journal Articles: Regenerative Medicine

BACKGROUND: IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents.

METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts …