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GSBS Student Publications

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Articles 1 - 30 of 32

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Dynamic Control Of Dendritic Mrna Expression By Cnot7 Regulates Synaptic Efficacy And Higher Cognitive Function, Rhonda L. Mcfleder, Fernanda Mansur, Joel D. Richter Jul 2017

Dynamic Control Of Dendritic Mrna Expression By Cnot7 Regulates Synaptic Efficacy And Higher Cognitive Function, Rhonda L. Mcfleder, Fernanda Mansur, Joel D. Richter

GSBS Student Publications

Translation of mRNAs in dendrites mediates synaptic plasticity, the probable cellular basis of learning and memory. Coordination of translational inhibitory and stimulatory mechanisms, as well as dendritic transport of mRNA, is necessary to ensure proper control of this local translation. Here, we find that the deadenylase CNOT7 dynamically regulates dendritic mRNA translation and transport, as well as synaptic plasticity and higher cognitive function. In cultured hippocampal neurons, synaptic stimulation induces a rapid decrease in CNOT7, which, in the short-term, results in poly(A) tail lengthening of target mRNAs. However, at later times following stimulation, decreased poly(A) and dendritic localization ...


Endoplasmic Reticulum Stress-Induced Hepatocellular Death Pathways Mediate Liver Injury And Fibrosis Via Stimulator Of Interferon Genes., Arvin Iracheta-Vellve, Jan Petrasek, Benedek Gyongyosi, Abhishek Satishchandran, Patrick Lowe, Karen Kodys, Donna Catalano, Charles D. Calenda, Evelyn A. Kurt-Jones, Kate A. Fitzgerald, Gyongyi Szabo Nov 2016

Endoplasmic Reticulum Stress-Induced Hepatocellular Death Pathways Mediate Liver Injury And Fibrosis Via Stimulator Of Interferon Genes., Arvin Iracheta-Vellve, Jan Petrasek, Benedek Gyongyosi, Abhishek Satishchandran, Patrick Lowe, Karen Kodys, Donna Catalano, Charles D. Calenda, Evelyn A. Kurt-Jones, Kate A. Fitzgerald, Gyongyi Szabo

GSBS Student Publications

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbontetrachloride (CCl4) administration to WT ...


The Exocyst Subunit Sec6 Interacts With Assembled Exocytic Snare Complexes, Michelle L. Dubuke, Stephanie Maniatis, Scott A. Shaffer, Mary Munson Nov 2015

The Exocyst Subunit Sec6 Interacts With Assembled Exocytic Snare Complexes, Michelle L. Dubuke, Stephanie Maniatis, Scott A. Shaffer, Mary Munson

GSBS Student Publications

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein ...


Determination Of Fatty Acid Oxidation And Lipogenesis In Mouse Primary Hepatocytes, Thomas E. Akie, Marcus P. Cooper Aug 2015

Determination Of Fatty Acid Oxidation And Lipogenesis In Mouse Primary Hepatocytes, Thomas E. Akie, Marcus P. Cooper

GSBS Student Publications

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis ...


Tailor: A Computational Framework For Detecting Non-Templated Tailing Of Small Silencing Rnas, Min-Te Chou, Bo W. Han, Chiung-Po Hsiao, Phillip D. Zamore, Zhiping Weng, Jui-Hung Hung May 2015

Tailor: A Computational Framework For Detecting Non-Templated Tailing Of Small Silencing Rnas, Min-Te Chou, Bo W. Han, Chiung-Po Hsiao, Phillip D. Zamore, Zhiping Weng, Jui-Hung Hung

GSBS Student Publications

Small silencing RNAs, including microRNAs, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), have been shown to play important roles in fine-tuning gene expression, defending virus and controlling transposons. Loss of small silencing RNAs or components in their pathways often leads to severe developmental defects, including lethality and sterility. Recently, non-templated addition of nucleotides to the 3' end, namely tailing, was found to associate with the processing and stability of small silencing RNAs. Next Generation Sequencing has made it possible to detect such modifications at nucleotide resolution in an unprecedented throughput. Unfortunately, detecting such events from millions of short ...


An Optimized Kit-Free Method For Making Strand-Specific Deep Sequencing Libraries From Rna Fragments, Erin E. Heyer, Hakan Ozadam, Emiliano P. Ricci, Can Cenik, Melissa J. Moore Jan 2015

An Optimized Kit-Free Method For Making Strand-Specific Deep Sequencing Libraries From Rna Fragments, Erin E. Heyer, Hakan Ozadam, Emiliano P. Ricci, Can Cenik, Melissa J. Moore

GSBS Student Publications

Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library ...


Direct Interactions Promote Eviction Of The Sir3 Heterochromatin Protein By The Swi/Snf Chromatin Remodeling Enzyme, Benjamin J. Manning, Craig L. Peterson Dec 2014

Direct Interactions Promote Eviction Of The Sir3 Heterochromatin Protein By The Swi/Snf Chromatin Remodeling Enzyme, Benjamin J. Manning, Craig L. Peterson

GSBS Student Publications

Heterochromatin is a specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. Despite its globally repressive role, heterochromatin must also be dynamic, allowing for its repair and replication. In budding yeast, heterochromatin formation requires silent information regulators (Sirs) Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating-type loci. Previously, we found that the SWI/SNF chromatin remodeling enzyme can catalyze the ATP-dependent eviction of Sir3p from recombinant nucleosomal arrays, and this activity enhances early steps of recombinational repair in vitro. Here, we show that the ATPase subunit of ...


The Middle X Residue Influences Cotranslational N-Glycosylation Consensus Site Skipping, Heidi Malaby, William R. Kobertz Aug 2014

The Middle X Residue Influences Cotranslational N-Glycosylation Consensus Site Skipping, Heidi Malaby, William R. Kobertz

GSBS Student Publications

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X not equal P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic ...


A Co-Crispr Strategy For Efficient Genome Editing In Caenorhabditis Elegans, Heesun Kim, Takao Ishidate, Krishna S. Ghanta, Meetu Seth, Darryl Conte Jr., Masaki Shirayama, Craig C. Mello Aug 2014

A Co-Crispr Strategy For Efficient Genome Editing In Caenorhabditis Elegans, Heesun Kim, Takao Ishidate, Krishna S. Ghanta, Meetu Seth, Darryl Conte Jr., Masaki Shirayama, Craig C. Mello

GSBS Student Publications

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new ...


Modulation Of Frustration In Folding By Sequence Permutation, Robert P. Nobrega, Karunesh Arora, Sagar V. Kathuria, Rita Graceffa, Raul A. Barrea, Liang Guo, Srinivas Chakravarthy, Osman Bilsel, Thomas C. Irving, Charles L. Brooks 3rd, C. Robert Matthews Jul 2014

Modulation Of Frustration In Folding By Sequence Permutation, Robert P. Nobrega, Karunesh Arora, Sagar V. Kathuria, Rita Graceffa, Raul A. Barrea, Liang Guo, Srinivas Chakravarthy, Osman Bilsel, Thomas C. Irving, Charles L. Brooks 3rd, C. Robert Matthews

GSBS Student Publications

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively ...


Rictor/Mtorc2 Loss In The Myf5 Lineage Reprograms Brown Fat Metabolism And Protects Mice Against Obesity And Metabolic Disease, Chien-Min Hung, Camila Martinez Calejman, Joan Sanchez-Gurmaches, Huawei Li, Clary B. Clish, Simone Hettmer, Amy J. Wagers, David A. Guertin Jul 2014

Rictor/Mtorc2 Loss In The Myf5 Lineage Reprograms Brown Fat Metabolism And Protects Mice Against Obesity And Metabolic Disease, Chien-Min Hung, Camila Martinez Calejman, Joan Sanchez-Gurmaches, Huawei Li, Clary B. Clish, Simone Hettmer, Amy J. Wagers, David A. Guertin

GSBS Student Publications

The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2) and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and ...


Angiomotins Link F-Actin Architecture To Hippo Pathway Signaling, Sebastian Mana-Capelli, Murugan Paramasivam, Shubham Dutta, Dannel Mccollum May 2014

Angiomotins Link F-Actin Architecture To Hippo Pathway Signaling, Sebastian Mana-Capelli, Murugan Paramasivam, Shubham Dutta, Dannel Mccollum

GSBS Student Publications

The Hippo pathway regulates the transcriptional coactivator YAP to control cell proliferation, organ size, and stem cell maintenance. Multiple factors, such as substrate stiffness, cell density, and G protein-coupled receptor signaling, regulate YAP through their effects on the F-actin cytoskeleton, although the mechanism is not known. Here we show that angiomotin proteins (AMOT130, AMOTL1, and AMOTL2) connect F-actin architecture to YAP regulation. First, we show that angiomotins are required to relocalize YAP to the cytoplasm in response to various manipulations that perturb the actin cytoskeleton. Second, angiomotins associate with F-actin through a conserved F-actin-binding domain, and mutants defective for F-actin ...


An Improved Predictive Recognition Model For Cys(2)-His(2) Zinc Finger Proteins, Ankit Gupta, Ryan G. Christensen, Heather A. Bell, Mathew Goodwin, Ronak Y. Patel, Manishi Pandey, Metewo Selase Enuameh, Amy L. Rayla, Cong Zhu, Stacey Thibodeau-Beganny, Michael H. Brodsky, J. Keith Joung, Scot A. Wolfe, Gary D. Stormo Apr 2014

An Improved Predictive Recognition Model For Cys(2)-His(2) Zinc Finger Proteins, Ankit Gupta, Ryan G. Christensen, Heather A. Bell, Mathew Goodwin, Ronak Y. Patel, Manishi Pandey, Metewo Selase Enuameh, Amy L. Rayla, Cong Zhu, Stacey Thibodeau-Beganny, Michael H. Brodsky, J. Keith Joung, Scot A. Wolfe, Gary D. Stormo

GSBS Student Publications

Cys(2)-His(2) zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ...


Folding Of The Rna Recognition Motif (Rrm) Domains Of The Amyotrophic Lateral Sclerosis (Als)-Linked Protein Tdp-43 Reveals An Intermediate State, Brian C. Mackness, Meme T. Tran, Shannan P. Mcclain, C. Robert Matthews, Jill A. Zitzewitz Mar 2014

Folding Of The Rna Recognition Motif (Rrm) Domains Of The Amyotrophic Lateral Sclerosis (Als)-Linked Protein Tdp-43 Reveals An Intermediate State, Brian C. Mackness, Meme T. Tran, Shannan P. Mcclain, C. Robert Matthews, Jill A. Zitzewitz

GSBS Student Publications

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that ...


Functional Overlap Among Distinct G1/S Inhibitory Pathways Allows Robust G1 Arrest By Yeast Mating Pheromones, Patricia A. Pope, Peter M. Pryciak Dec 2013

Functional Overlap Among Distinct G1/S Inhibitory Pathways Allows Robust G1 Arrest By Yeast Mating Pheromones, Patricia A. Pope, Peter M. Pryciak

GSBS Student Publications

In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This ...


Sub-Millisecond Time-Resolved Saxs Using A Continuous-Flow Mixer And X-Ray Micro-Beam, Rita Graceffa, Robert P. Nobrega, Raul Barrea, Sagar V. Kathuria, Srinivas Chakravarthy, Osman Bilsel, Thomas Irving Nov 2013

Sub-Millisecond Time-Resolved Saxs Using A Continuous-Flow Mixer And X-Ray Micro-Beam, Rita Graceffa, Robert P. Nobrega, Raul Barrea, Sagar V. Kathuria, Srinivas Chakravarthy, Osman Bilsel, Thomas Irving

GSBS Student Publications

Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam ...


Latent Effects Of Hsp90 Mutants Revealed At Reduced Expression Levels, Li Jiang, Parul Mishra, Ryan T. Hietpas, Konstantin B. Zeldovich, Daniel N. A. Bolon Jun 2013

Latent Effects Of Hsp90 Mutants Revealed At Reduced Expression Levels, Li Jiang, Parul Mishra, Ryan T. Hietpas, Konstantin B. Zeldovich, Daniel N. A. Bolon

GSBS Student Publications

In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects ...


Crosstalk Between Casein Kinase Ii And Ste20-Related Kinase Nak1, Lubos Cipak, Sneha Gupta, Iva Rajovic, Quan-Wen Jin, Dorothea Anrather, Gustav Ammerer, Dannel Mccollum, Juraj Gregan Mar 2013

Crosstalk Between Casein Kinase Ii And Ste20-Related Kinase Nak1, Lubos Cipak, Sneha Gupta, Iva Rajovic, Quan-Wen Jin, Dorothea Anrather, Gustav Ammerer, Dannel Mccollum, Juraj Gregan

GSBS Student Publications

Although the sterile 20 (Ste20) serine/threonine protein kinase was originally identified as a component of the S. cerevisiae mating pathway, it has homologs in higher eukaryotes and is part of a larger family of Ste20-like kinases. Ste20-like kinases are involved in multiple cellular processes, such as cell growth, morphogenesis, apoptosis and immune response. Carrying out such a diverse array of biological functions requires numerous regulatory inputs and outputs in the form of protein-protein interactions and post-translational modifications. Hence, a thorough knowledge of Ste20-like kinase binding partners and phosphorylation sites will be essential for understanding the various roles of these ...


Screening For Melanoma Modifiers Using A Zebrafish Autochthonous Tumor Model, Sharanya Iyengar, Yariv Houvras, Craig J. Ceol Nov 2012

Screening For Melanoma Modifiers Using A Zebrafish Autochthonous Tumor Model, Sharanya Iyengar, Yariv Houvras, Craig J. Ceol

GSBS Student Publications

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using ...


Systematic Dissection Of Roles For Chromatin Regulators In A Yeast Stress Response, Assaf Weiner, Hsiuyi V. Chen, Chih Long Liu, Ayelet Rahat, Avital Klien, Luis Soares, Mohanram Gudipati, Jenna Pfeffner, Aviv Regev, Stephen Buratowski, Jeffrey A. Pleiss, Nir Friedman, Oliver J. Rando Jul 2012

Systematic Dissection Of Roles For Chromatin Regulators In A Yeast Stress Response, Assaf Weiner, Hsiuyi V. Chen, Chih Long Liu, Ayelet Rahat, Avital Klien, Luis Soares, Mohanram Gudipati, Jenna Pfeffner, Aviv Regev, Stephen Buratowski, Jeffrey A. Pleiss, Nir Friedman, Oliver J. Rando

GSBS Student Publications

Packaging of eukaryotic genomes into chromatin has wide-ranging effects on gene transcription. Curiously, it is commonly observed that deletion of a global chromatin regulator affects expression of only a limited subset of genes bound to or modified by the regulator in question. However, in many single-gene studies it has become clear that chromatin regulators often do not affect steady-state transcription, but instead are required for normal transcriptional reprogramming by environmental cues. We therefore have systematically investigated the effects of 83 histone mutants, and 119 gene deletion mutants, on induction/repression dynamics of 170 transcripts in response to diamide stress in ...


Regulation Of Exocytosis By The Exocyst Subunit Sec6 And The Sm Protein Sec1, Francesca Morgera, Margaret R. Sallah, Michelle L. Dubuke, Pallavi Gandhi, Daniel N. Brewer, Chavela M. Carr, Mary Munson Jan 2012

Regulation Of Exocytosis By The Exocyst Subunit Sec6 And The Sm Protein Sec1, Francesca Morgera, Margaret R. Sallah, Michelle L. Dubuke, Pallavi Gandhi, Daniel N. Brewer, Chavela M. Carr, Mary Munson

GSBS Student Publications

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 ...


Conformational Lability In The Class Ii Mhc 3-10 Helix And Adjacent Extended Strand Dictate Hla-Dm Susceptibility And Peptide Exchange, Corrie A. Painter, Maria P. Negroni, Kathy A. Kellersberger, Zarixia Zavala-Ruiz, James E. Evans, Lawrence J. Stern Nov 2011

Conformational Lability In The Class Ii Mhc 3-10 Helix And Adjacent Extended Strand Dictate Hla-Dm Susceptibility And Peptide Exchange, Corrie A. Painter, Maria P. Negroni, Kathy A. Kellersberger, Zarixia Zavala-Ruiz, James E. Evans, Lawrence J. Stern

GSBS Student Publications

HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the ...


Chitosan But Not Chitin Activates The Inflammasome By A Mechanism Dependent Upon Phagocytosis, Chelsea L. Bueter, Chrono K. Lee, Vijay A. K. Rathinam, Gloria J. Healy, Christopher H. Taron, Charles A. Specht, Stuart M. Levitz Oct 2011

Chitosan But Not Chitin Activates The Inflammasome By A Mechanism Dependent Upon Phagocytosis, Chelsea L. Bueter, Chrono K. Lee, Vijay A. K. Rathinam, Gloria J. Healy, Christopher H. Taron, Charles A. Specht, Stuart M. Levitz

GSBS Student Publications

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1beta. We found that although chitosan was a potent NLRP3 inflammasome ...


Argonaute Protein Identity And Pairing Geometry Determine Cooperativity In Mammalian Rna Silencing, Jennifer A. Broderick, William E. Salomon, Sean P. Ryder, Neil Aronin, Phillip D. Zamore Oct 2011

Argonaute Protein Identity And Pairing Geometry Determine Cooperativity In Mammalian Rna Silencing, Jennifer A. Broderick, William E. Salomon, Sean P. Ryder, Neil Aronin, Phillip D. Zamore

GSBS Student Publications

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs ...


Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven F. Trueman, Elisabet C. Mandon, Reid Gilmore Sep 2011

Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven F. Trueman, Elisabet C. Mandon, Reid Gilmore

GSBS Student Publications

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in ...


Sorting Of Drosophila Small Silencing Rnas Partitions Microrna* Strands Into The Rna Interference Pathway, Megha Ghildiyal, Jia Xu, Herve Seitz, Zhiping Weng, Phillip D. Zamore Jan 2010

Sorting Of Drosophila Small Silencing Rnas Partitions Microrna* Strands Into The Rna Interference Pathway, Megha Ghildiyal, Jia Xu, Herve Seitz, Zhiping Weng, Phillip D. Zamore

GSBS Student Publications

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character-as is found in small interfering RNAs (siRNAs)-directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient ...


Discovery Of A Novel Activator Of Kcnq1-Kcne1 K Channel Complexes., Karen Mruk, William R. Kobertz Jan 2009

Discovery Of A Novel Activator Of Kcnq1-Kcne1 K Channel Complexes., Karen Mruk, William R. Kobertz

GSBS Student Publications

KCNQ1 voltage-gated K(+) channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K(+) channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus ...


Model For The Peptide-Free Conformation Of Class Ii Mhc Proteins, Corrie A. Painter, Anthony Cruz, Gustavo E. Lopez, Lawrence J. Stern, Zarixia Zavala-Ruiz Jun 2008

Model For The Peptide-Free Conformation Of Class Ii Mhc Proteins, Corrie A. Painter, Anthony Cruz, Gustavo E. Lopez, Lawrence J. Stern, Zarixia Zavala-Ruiz

GSBS Student Publications

BACKGROUND: Major histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive.

METHODOLOGY/PRINCIPAL FINDINGS: Here we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the alpha50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe alpha54 ...


The Role Of Ire1alpha In The Degradation Of Insulin Mrna In Pancreatic Beta-Cells, Kathryn L. Lipson, Rajarshi Ghosh, Fumihiko Urano Feb 2008

The Role Of Ire1alpha In The Degradation Of Insulin Mrna In Pancreatic Beta-Cells, Kathryn L. Lipson, Rajarshi Ghosh, Fumihiko Urano

GSBS Student Publications

BACKGROUND:The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1alpha is a central component of the UPR. In pancreatic beta-cells, IRE1alpha also functions in the regulation of insulin biosynthesis.

PRINCIPAL FINDINGS:Here we report that hyperactivation of IRE1alpha caused by chronic high glucose treatment or IRE1alpha overexpression leads to insulin mRNA degradation in pancreatic beta-cells. Inhibition of IRE1alpha signaling using its dominant negative form prevents insulin ...


Designing Sirna That Distinguish Between Genes That Differ By A Single Nucleotide, Dianne S. Schwarz, Hongliu Ding, Lori A. Kennington, Jessica T. Moore, Janell M. Schelter, Julja Burchard, Peter S. Linsley, Neil Aronin, Zuoshang Xu, Phillip D. Zamore Sep 2006

Designing Sirna That Distinguish Between Genes That Differ By A Single Nucleotide, Dianne S. Schwarz, Hongliu Ding, Lori A. Kennington, Jessica T. Moore, Janell M. Schelter, Julja Burchard, Peter S. Linsley, Neil Aronin, Zuoshang Xu, Phillip D. Zamore

GSBS Student Publications

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide ...