DOCSLIB.ORG

Encounter with Antigen-Specific Primed CD4 T Cells Promotes MHC Class II Degradation in Dendritic Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Encounter with Antigen-Specific Primed CD4 T Cells Promotes MHC Class II Degradation in Dendritic Cells Encounter with antigen-specific primed CD4 T cells promotes MHC class II degradation in dendritic cells Kazuyuki Furutaa, Satoshi Ishidob, and Paul A. Rochea,1 aExperimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and bLaboratory of Infectious Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved October 12, 2012 (received for review August 9, 2012) Major histocompatibility complex class II molecules (MHC-II) on murine APCs and acquired by mouse CD4 T cells (14); however, antigen presenting cells (APCs) engage the TCR on antigen-specific this acquisition is antigen independent. Huang et al. have shown CD4 T cells, thereby providing the specificity required for T cell that MHC-I can be transferred to antigen-specific CD8 T cells priming and the induction of an effective immune response. In this after conjugate formation (15); however, this process merely study, we have asked whether antigen-loaded dendritic cells (DCs) transfers intact MHC protein from the APC to the antigen- fi that have been in contact with antigen-specific CD4 T cells retain the speci c T-cell and promotes T-cell fratricide (15). Prolonged interactions of antigen-loaded DCs with antigen- ability to stimulate additional naïve T cells. We show that encounter fi with antigen-specific primed CD4 T cells induces the degradation of speci c T cells are required to initiate naïve CD4 T-cell activa- surface MHC-II in antigen-loaded DCs and inhibits the ability of tion and proliferation (16, 17). This process is tightly regulated to allow the effective initiation of an immune response. However, these DCs to stimulate additional naïve CD4 T cells. Cross-linking once this response is established, there could be mechanisms with MHC-II mAb as a surrogate for T-cell engagement also inhibits that limit overstimulation of additional antigen-specificTcells, APC function and induces MHC-II degradation by promoting the a process that could initiate a pathological inflammatory cascade. clustering of MHC-II present in lipid raft membrane microdomains, In this study, we have investigated the function of DCs after a process that leads to MHC-II endocytosis and degradation in lyso- engagement with antigen-specific CD4 T cells. We find that an fi somes. Encounter of DCs with antigen-speci c primed T cells or encounter of antigen-experienced T cells with antigen-bearing engagement of MHC-II with antibodies promotes the degradation APCs promotes the down-regulation of surface MHC-II ex- of both immunologically relevant and irrelevant MHC-II molecules. pression in DCs and renders these DCs incapable of stimulating IMMUNOLOGY These data demonstrate that engagement of MHC-II on DCs after additional naïve CD4 T cells. Essentially identical results are encounter with antigen-specific primed CD4 T cells promotes the obtained when MHC-II was cross-linked with MHC-II mAb, down-regulation of cell surface MHC-II in DCs, thereby attenuating a process that results in lipid raft-dependent MHC-II clustering, additional activation of naïve CD4 T cells by these APCs. endocytosis, and lysosomal degradation. Finally, we show that the down-regulation of MHC-II by T-cell engagement or MHC- T-cell activation | protein aggregation II cross-linking severely limits the ability of the DC to stimulate even unrelated naïve CD4 T cells. Our data thus demonstrate endritic cells (DCs) are professional antigen presenting cells that one consequence of CD4 T-cell recognition of antigen- (APCs) that function to prime naïve CD4 T cells through bearing APCs is down-regulation of surface pMHC-II by endo- D cytosis and lysosomal degradation, thereby limiting the ability of cell surface major histocompatibility complex class II (MHC-II) fi molecules. Immature DCs have high potency of antigen capture these APCs to interact with additional antigen-speci c T cells. and processing. After capturing antigen, DCs migrate to draining Results and Discussion lymph nodes, where they mature and express peptide-loaded MHC-II (pMHC-II) on their cell surface (1, 2). The pMHC-II on Encounter with Antigen-Specific CD4 T Cells Inhibits Subsequent MHC- the surface of APCs then “presents” the expressed antigenic II–Restricted APC Function of DCs. Although it is well known that peptide to naïve antigen-specific CD4 T cells, a process that DCs can efficiently prime naïve CD4 T cells, it remains to be results in the proliferation and activation of CD4 T cells (3). seen whether these DCs are capable of activating additional In APCs, cell surface MHC-II expression is tightly regulated naïve CD4 T cells of the same (or differing) specificity after they have completed their task of T-cell priming. To examine this and is important for the generation and propagation of an im- k mune response (4). Newly synthesized MHC-II forms a complex question, we have pretreated HEL-pulsed DCs with I-A - – with a chaperone protein termed the invariant chain (Ii) in the HEL46–61 restricted 3A9 CD4 T cells before adding additional fl endoplasmic reticulum. The MHC-II–Ii complex is transported carboxy uorescein succinimidyl ester (CFSE)-tagged naïve 3A9 to cell surface, internalized, and targeted to the antigen pre- T cells to the culture. Preincubation of the DCs with naïve senting compartment where Ii is degraded and MHC-II loads control or 3A9 T cells had no effect on the ability of the DCs to A with antigenic peptides (5). Peptide-loaded MHC-II is then simulate additional naïve 3A9 T cells (Fig. 1 ). Surprisingly, transported to the cell surface to present antigenic peptides to preincubation of the DCs with previously primed 3A9 CD4 CD4 T cells. Once at the cell surface, pMHC can internalize and T cells, but not control T cells, almost completely prevented the enter the endocytic pathway. Some fraction of internalized subsequent proliferation of CFSE-labeled naïve 3A9 T cells by A A pMHC-II is then recycled back to cell surface (6–8) while an- these DCs (Fig. 1 and Fig. S1 ). To rule out the possibility that other fraction is ubiquitinated by the E3 ubiquitin ligase March-I the proliferation of the T cells in the preculture affected naïve and targeted for degradation in lysosomes (9, 10). CD4 T-cell proliferation, the entire preculture was irradiated There are many reports demonstrating that upon conjugate formation between antigen-specific CD4 T cells with antigen- bearing APCs, the TCR internalizes and is degraded in the T Author contributions: K.F. and P.A.R. designed research; K.F. performed research; S.I. con- cell, a process that is thought to limit T-cell activation by APCs tributed new reagents/analytic tools; K.F. and P.A.R. analyzed data; and K.F. and P.A.R. (11, 12). This process can be mimicked by TCR cross-linking wrote the paper. with mAb (13), leading to the widely held belief that in vivo TCR The authors declare no conflict of interest. cross-linking by APC ligands promotes TCR endocytosis and This article is a PNAS Direct Submission. degradation. By contrast, the fate of the stimulatory pMHC-II 1To whom correspondence should be addressed. E-mail: [email protected]. complexes on APCs after an encounter with antigen-specific This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CD4 T cells has not been followed. MHC-II can be shed from 1073/pnas.1213868109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213868109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Preculture with antigen-specific T cells inhibits subsequent naïve T-cell proliferation by DCs. (A) HEL-loaded DCs were pretreated alone (no T cells), with naïve or primed control CD4 T cells − k (3A9 tg ), or with naïve or primed I-A -HEL46–61– specific 3A9 CD4 T cells (3A9 tg+), for 4 h at 1:1 ratio. After various treatments, naïve CFSE-labeled 3A9 CD4 T cells were added to the culture at a 1:10 ratio (DC:naïve T-cell), and naïve 3A9 CD4 T-cell pro- liferation was measured 48 h later by FACS analysis. (B) HEL-loaded DCs were preincubated alone (no T cells) or with primed 3A9 tg− or primed 3A9 tg+ T cells for 4 h. The entire pretreated culture was ir- radiated with 3,000 rad, washed, and then added directly to CFSE-labeled 3A9 T cells. In each experi- ment the division index was calculated by using FlowJo software. The division index under each con- dition was expressed relative to that culture condi- tion in which CD4 T cells were not added. The data shown are the mean ± SD from three independent experiments. *P < 0.05 (relative to no T cells control). before the addition of CFSE-labeled naïve 3A9 T cells. Once Cross-Linking of MHC-II Promotes Endocytosis and Lysosomal again, preculture with antigen-specific T cells prevented sub- Degradation of Surface MHC-II in DCs. The APC-mediated down- sequent naïve CD4 T-cell proliferation, whereas preculture with regulation of the TCR upon T-cell:APC conjugate formation can nonspecific T cells did not (Fig. 1B). Lastly, we purified the DCs be mimicked by TCR cross-linking using anti-TCR mAb (13). To after the preculture period with nonspecific or antigen-specificT mimic the engagement of MHC-II with ligand(s) on T cells, we cells, and even these purified DCs were unable to stimulate naïve cross-linked MHC-II on the surface of DCs by using MHC-II 3A9 T-cell proliferation (Fig. S1B), demonstrating that the en- mAb.
Load more

Recommended publications
  • Biotinylated Standards Kit
    4006050C 5/24/99 8:35 AM Page i Biotinylated Standards Kit Catalog Numbers 161-0307 161-0308 161-0312 161-0313 161-0321 161-0322 4006050C 5/24/99 8:35 AM Page i Table of Contents Section 1 Introduction .......................................................................... 1 Section 2 Specifications ........................................................................ 3 Section 3 Safety Instructions................................................................ 4 Section 4 Solutions ................................................................................ 5 4.1 Solutions for Electrophoresis Gels ......................................... 5 4.2 Solutions for Nitrocellulose Membranes................................ 5 Section 5 Assay Procedure ................................................................... 7 5.1 General Recommendations..................................................... 7 5.2 Protocol for SDS-PAGE Electrophoresis............................... 7 5.3 Protocol for Immune Detection .............................................. 8 Section 6 Troubleshooting Guide ........................................................ 9 6.1 Activity Test for Reagents...................................................... 9 6.2 No Reaction or Weak Color Development............................. 9 6.3 No Color in Standards, Good Color in Sample ...................... 10 6.4 No Color in Sample, Good Color in Standards ...................... 11 6.5 High Background...................................................................
    [Show full text]
  • Biotinylation of Protein for Immobilization Onto Streptavidin
    TECHNICAL NOTE 28 Biotinylation of proteins for immobilization onto Streptavidin biosensors Overview • Protein to be biotinylated (requires at least 50 µg total at a concentration of at least 100 µg/mL) The interaction between streptavidin and biotin is widely used as a system for the rapid, stable and irreversible non-covalent • (Optional) Slide-A-Lyzer (Thermo, part no. 66370) binding of biological molecules. The Octet® platform’s Strepta- vidin biosensors have been developed for the immobilization Protein sample preparation of biotinylated ligands for both quantitation and kinetic applica- • Ensure that the protein is carrier-protein free (for antibodies tions. The first protein immobilized onto the Streptavidin biosen- containing carrier protein, see ForteBio Technical Note 11.) sors must be biotinylated prior to assaying on the Octet system. • Ensure that the protein to be biotinylated is not in a buffer This technical note supersedes ForteBio Technical Notes 6 containing primary amines (i.e., Tris or glycine). If protein is in (Biotinylation of Protein for Immobilization onto Streptavidin a buffer containing primary amines, exchange into PBS either K Biosensors) and 12 (Biotinylating Very Small Quantities of by dialysis or desalting spin columns. The protein concentra- Protein for Immobilization onto Streptavidin Biosensors). It tion is recommended to be at least 1 mg/mL for this process. provides guidelines for biotinylating a protein of interest utilizing Pierce-ThermoFisher (piercenet.com) biotinylation PROTEIN SAMPLE PREPARATION BY DIALYSIS reagents for kinetic and quantitation assays with Streptavidin (Recommended for samples >0.5mL) biosensors on the Octet platform. 1 Dialyze each sample 1:1000 in 1X PBS using a Slide-A-Lyzer.
    [Show full text]
  • One-Step Antibody Biotinylation
    One-step Antibody Biotinylation Kit Order no. 130-093-385 Contents 1.2 Background information 1. Description The One-step Antibody Biotinylation Kit has been developed for the biotinylation of monoclonal antibodies for use in magnetic cell 1.1 Principle of the One-step Antibody Biotinylation Kit separation (with MACS® Technology) as well as fluorescent cell 1.2 Background information analysis. 1.3 Applications Once biotinylated, antibodies can be directly used to tag target cells. Subsequently, target cells can either be magnetically labeled using 1.4 Reagent requirements Anti-Biotin MicroBeads (# 130-090-485) or fluorescently stained 2. Protocol with a FITC-, PE-, or APC-conjugated anti-biotin antibody. After labeling with Anti-Biotin MicroBeads, target cells can be enriched 3. Examples of immunofluorescent staining with biotinylated according to the protocol in the Anti-Biotin MicroBeads data sheet. antibodies Anti-Biotin MicroBead 1. Description Biotinylated primary antibody This product is for research use only. Components 1 strip of 8 reaction wells, each containing Figure 1: Immunomagnetic labeling principle. lyophilized Biotinylation Mix. 1.3 Applications Capacity For 8 labeling reactions of up to 10 μg of antibody each. ● Biotinylation of monoclonal antibodies. Product format The labeling agent is supplied as a lyophilized 1.4 Reagent requirements powder and is optimally apportioned for the labeling of up to 10 μg of antibody per well. Each ● Antibody: The antibody to be biotinylated must be purified well can be cut from the strip for individual from azide, serum components, and other NH₂-containing use or multiple antibodies can be labeled molecules prior to biotinylation.
    [Show full text]
  • S1 Supporting Information for Macrophage Uptake of Janus Particles Depends Upon Janus Balance Yuan Gao and Yan Yu Department Of
    Supporting Information for Macrophage Uptake of Janus Particles Depends upon Janus Balance Yuan Gao and Yan Yu* Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States *Corresponding author: [email protected] Contents Experimental Section Supplementary Figures S1-S3 S1 Experimental Section 1. Cells and reagents Monodisperse silica particles were purchased from Spherotech (Lake Forest, IL). Sylgard 184 silicone elastomer kit was purchased from Dow Corning (Midland, MI). Pluronic F- 127, (3-Aminopropyl) triethoxysilane (APTES), biotin N-hydroxysuccinimide ester (biotin-NHS), bovine serum albumin (BSA) and immunoglobulin G (IgG) from rabbit serum were purchased from Sigma-Aldrich (St. Louis, MO). Biotinylated bovine serum albumin (BSA-biotin) was purchased from Thermo Scientific (Waltham, MA). Streptavidin (SA), streptavidin Alexa Fluor-568 conjugate, Fluo-4 acetoxymethyl ester (Fluo-4) and Alexa Fluor-568 succinimidyl ester were purchased from Life Technologies (Grand Island, NY). VivoTrack 680 NIR fluorescence imaging agent was purchased from Perkin Elmer (Waltham, MA). BSA-biotin Alexa Fluor-568 conjugate was synthesized from BSA-biotin and Alexa Fluor-568 succinimidyl ester. IgG was biotinylated with biotin-NHS. Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl (RhB- PE) were purchased from Avanti Polar Lipid (Alabaster, AL). RAW264.7 macrophage cells were purchased from ATCC (Manassas, VA). RAW264.7 macrophage cells were cultured in DMEM complete growth media supplemented with 10% fetal bovine serum (FBS), 0.11 mg/ml (1 mM) sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin. Ringer’s imaging buffer (pH = 7.2, 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM HEPES, 10 mM glucose) was used for all live-cell imaging.
    [Show full text]
  • Open Dissertation Sambeeta Das.Pdf
    The Pennsylvania State University The Graduate School Eberly College of Science DESIGNS FOR DIRECTING MOTION AT THE MICRO- AND NANOSCALE A Dissertation in Chemistry by Sambeeta Das © 2016 Sambeeta Das Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosphy August 2016 The dissertation of Sambeeta Das was reviewed and approved* by the following: Ayusman Sen Distinguished Professor of Chemistry Dissertation Advisor Chair of Committee Thomas E. Mallouk Evan Pugh Professor of Chemistry, Physics, Biochemistry and Molecular Biology Head of the Chemistry Department Associate Director, Penn State MRSEC Director, Center for Solar Nanomaterials John Badding Professor of Chemistry Associate Head for Equity and Diversity Director of Graduate Recruiting Darrell Velegol Distinguished Professor of Chemical Engineering *Signatures are on file in the Graduate School ii Abstract Motion is one of the defining characteristics of life. Inspired by the biological motors at the micron and nanoscale, scientists have developed artificial machines that self propel in solutions and are on the same length scale as biological motors. However, motion at such low length scales is fraught with many challenges. The focus of this thesis is on devising some field free and autonomous strategies for addressing those challenges. This dissertation starts with a literature review of the field with a discussion about the various self-propelled prevalent in the scientific community. Then the different challenges of motion at low Reynolds number and their applicability to the motors are discussed. One of the more popular categories of catalytic micromotors is Janus motors which move in a solution of hydrogen peroxide due to the catalytic activity of Platinum.
    [Show full text]
  • Airid, a Novel Proximity Biotinylation Enzyme, for Analysis of Protein
    TOOLS AND RESOURCES AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions Kohki Kido1, Satoshi Yamanaka1, Shogo Nakano2, Kou Motani3, Souta Shinohara1, Akira Nozawa1, Hidetaka Kosako3, Sohei Ito2, Tatsuya Sawasaki1* 1Division of Cell-Free Life Science, Proteo-Science Center, Matsuyama, Japan; 2Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan; 3Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan Abstract Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IkBa indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IkBa showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions. *For correspondence: [email protected] Introduction Competing interests: The Many cellular proteins function under the control of biological regulatory systems. Protein–protein authors declare that no interactions (PPIs) comprise part of the biological regulation system for proteins.
    [Show full text]
  • MM Biotinylation System for Mouse on Mouse IHC Staining Detection Kit Control Number: 902-MMBK-081315
    MM Biotinylation System For Mouse on Mouse IHC Staining Detection Kit Control Number: 902-MMBK-081315 Catalog Number: MMBK H Description: 25 ml Species Reactivity: N/A Intended Use: Storage and Stability: For Research Use Only. Not for use in diagnostic procedures. Store at 2ºC to 8ºC. Do not use after expiration date printed on vial. If reagents are stored under conditions other than those specified in the package insert, they must be Summary & Explanation: verified by the user. Diluted reagents should be used promptly; any remaining reagent MM Biotinylation Kit is a biotinylated detection specially formulated to eliminate should be stored at 2ºC to 8ºC. endogenous mouse IgG background staining when using a mouse primary antibody on Staining Protocol Recommendations: mouse tissue. Even the most difficult tissues such as lymphatic, lung and kidney tissues are virtually background free. Slide Preparation: (Paraffin-Embedded) 1. Cut tissues 4 microns thick. (Do not use adhesives in the water bath). When a mouse monoclonal antibody is desired for immunohistochemical detection on 2. Place the tissue sections on a positive-charged slide. murine tissues, the biotinylated secondary antibody used for detection will bind to the 3. Drain excess water off the slides primary antibody and will bind to endogenous mouse IgG in the tissue. Troublesome 4. Dry at 37ºC to 40ºC overnight. Then dry slides for 15-30 minutes at 60ºC. background staining may occur from bound mouse IgG. Preparation for the Biotinylation of a Primary Antibody: The majority of primary antibodies on the market are not biotinylated. It is very 1. Add the proper quantities of Diluent, Concentrated Primary Antibody, and expensive and time consuming to biotinylate an antibody.
    [Show full text]
  • The Evolving Capabilities of Enzyme-Mediated Proximity Labeling Ying Zhou1 and Peng Zou1,2
    Available online at www.sciencedirect.com ScienceDirect The evolving capabilities of enzyme-mediated proximity labeling Ying Zhou1 and Peng Zou1,2 e Abstract dynamic liquid-like condensates that form via liquid The subcellular organization of proteins and RNA molecules is liquid phase separation (LLPS) [8]. For example, stal- crucial for their proper functions. Over the past decade, both led translation-initiation causes mRNAs and mRNA- ligase-mediated and peroxidase-mediated proximity labeling binding proteins (RBPs) to assemble into stress gran- (PL) techniques have been developed to map biomolecules at ules (SGs), in a process of protecting cells from oxida- near-nanometer spatial resolution and subminute temporal tive damages or other cellular stress [9]. resolution. These methods are shedding light on the spatial arrangement of proteome and transcriptome in their native The subcellular proteome and transcriptome have been context. Here, we review the recent evolution and applications traditionally investigated by co-immunoprecipitation of PL techniques, compare and contrast the two classes of (co-IP) and biochemical fractionation. However, both methods, and highlight emerging trends and future methods require prior cell lysis, which is prone to losing opportunities. low-affinity and transient proteineprotein interactions. In addition, co-IP is limited by the availability of high- Addresses quality antibodies against the bait, while biochemical 1 College of Chemistry and Molecular Engineering, Synthetic and fractionation often suffers from incomplete purification. Functional Biomolecules Center, Beijing National Laboratory for Mo- lecular Sciences, Key Laboratory of Bioorganic Chemistry and Mo- For example, the transcriptomic profiling of isolated lecular Engineering of Ministry of Education, Peking University, Beijing, mitochondria has identified abundant contaminations 100871, China from the cytoplasm [10].
    [Show full text]
  • Bioinspired Polymeric High‐Aspect‐Ratio Particles with Asymmetric Janus Functionalities
    RESEARCH ARTICLE www.advnanobiomedres.com Bioinspired Polymeric High-Aspect-Ratio Particles with Asymmetric Janus Functionalities Joel A. Finbloom, Yiqi Cao, and Tejal A. Desai* and particles have been engineered with Polymeric particles with intricate morphologies and properties have been diverse morphologies and physicochemical developed based on bioinspired designs for applications in regenerative medi- properties for applications such as tissue [7,8] cine, tissue engineering, and drug delivery. However, the fabrication of particles engineering and drug delivery. However, the design of polymeric particles with asymmetric functionalities remains a challenge. Janus polymeric particles with asymmetric distributions of functional- are an emerging class of materials with asymmetric functionalities; however, they ities remains a challenge. Many examples are predominantly spherical in morphology, made from nonbiocompatible of materials with asymmetric regions materials, and made using specialized fabrication techniques. Herein non- of functionality are found throughout [9] spherical Janus particles inspired by high-aspect-ratio filamentous bacteriophage natural systems. Proteins have regiospe- fi are fabricated using polycaprolactone polymers and standard methods. Janus ci c functionalities and biochemical proper- ties, which lead to controlled assembly high-aspect-ratio particles (J-HARPs) are fabricated with a nanotemplating dynamics, enzymatic active sites, and spe- technique to create branching morphologies selectively at one edge of the cificbindingdomainsofantibodies.[10,11] particle. J-HARPs are fabricated with maleimide handles and modified with Viruses such as filamentous bacteriophage biomolecules such as proteins and biotin. Regioselective modification is observed contain multivalent displays of peptides at the tips of J-HARPs, likely due to the increased surface area of the branching on one end of the virus particle to enable directional binding to targets.[12] Such regions.
    [Show full text]
  • Aggregation of Biotinylated Polymeric Microspheres Induced by Interaction with Avidin*
    Pure Appl. Chem., Vol. 79, No. 9, pp. 1575–1582, 2007. doi:10.1351/pac200779091575 © 2007 IUPAC Aggregation of biotinylated polymeric microspheres induced by interaction with avidin* Qingbin Meng1, Zhanyong Li1, Gang Li1, Xu Zhang1, Yingli An1, and X. X. Zhu1,2,‡ 1Key Laboratory of Functional Polymer Materials, Institute of Polymer Chemistry, Nankai University, Tianjin, 300071, China; 2Department of Chemistry, University of Montréal, C.P. 6128, succ. Centre-ville, Montréal, QC, H3C 3J7, Canada Abstract: Monodisperse biotinylated poly(styrene-co-N-acryloxysuccinimide) microspheres were synthesized in aqueous solutions with a two-step method. Upon the addition of avidin solutions of different concentrations in phosphate buffer into the dispersed biotinylated microspheres, the microspheres aggregated rapidly due to the high binding affinity between biotin and avidin. The hydrodynamic diameter of the aggregates and the aggregation rate ob- served at given time intervals increased with increasing concentration of avidin. The compo- sition of the microspheres and the incorporation of biotin were evidenced by Fourier trans- form infrared spectroscopy. The morphology, size distribution, and aggregation of the microspheres were studied by techniques such as scanning electron microscopy and dynamic light scattering. Keywords: polymer microspheres; biotinylation; emulsion polymerization; bioconjugation; aggregation. INTRODUCTION The detection and analysis of biological molecules have attracted much attention over the past few years. The rapid and
    [Show full text]
  • Dissecting Bcr-Abl Variant Signaling Pathways Using
    DISSECTING BCR-ABL VARIANT SIGNALING PATHWAYS USING NOVEL INTERACTOME IDENTIFICATION STRATEGIES By Jevon A. Cutler A dissertation submitted to The Johns Hopkins University in conformity with the requirement of the degree of Doctor of Philosophy Baltimore, MD May 2018 © 2018 Jevon A. Cutler All Rights Reserved ABSTRACT Cell signaling is an essential function of cells and tissues. Understanding cell signaling necessitates technologies that can identify protein-protein interactions as well as post translational modifications to proteins within protein complexes. The goals of this study are (1) to understand how BCR-ABL variants differentially signal to produce different clinical/experimental phenotypes and (2) to develop novel interactome detection strategies to understand signaling. This dissertation describes an integrated approach of the use of proximity dependent labeling protein-protein interaction analysis assays coupled with global phosphorylation analysis to investigate the differences in signaling between two variants the oncogenic fusion protein, BCR-ABL. Two major types of leukemogenic BCR-ABL fusion proteins are p190BCR-ABL and p210BCR-ABL. Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. Our findings suggest that p190BCR-ABL and p210BCR-ABL differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210BCR-ABL, we observed an increased engagement of molecules active proximal to the membrane and in the case of p190BCR-ABL, an engagement of molecules of the cytoskeleton.
    [Show full text]
  • Examination of Herpesvirus Entry Glycoprotein Interactions Using Proximity Biotinylation
    DePaul University Via Sapientiae College of Science and Health Theses and Dissertations College of Science and Health Summer 8-22-2014 Examination of herpesvirus entry glycoprotein interactions using proximity biotinylation Michelle Lajko DePaul University, [email protected] Follow this and additional works at: https://via.library.depaul.edu/csh_etd Recommended Citation Lajko, Michelle, "Examination of herpesvirus entry glycoprotein interactions using proximity biotinylation" (2014). College of Science and Health Theses and Dissertations. 78. https://via.library.depaul.edu/csh_etd/78 This Thesis is brought to you for free and open access by the College of Science and Health at Via Sapientiae. It has been accepted for inclusion in College of Science and Health Theses and Dissertations by an authorized administrator of Via Sapientiae. For more information, please contact [email protected]. Examination of herpesvirus entry glycoprotein interactions using proximity biotinylation A Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2014 By: Michelle Lajko Advisor: Dr. Sarah Connolly Department of Biological Sciences College of Science and Health DePaul University Chicago, IL Acknowledgements I would like to thank my advisor and mentor, Dr. Sarah Connolly for taking a graduate student on in a stressful first year. She supported me through my Master’s encouraging me to become a better critical thinker and therefore a better scientist. I also want to thank the other lab members: Alexander F. Haddad and Carolyn A. Robinson. Without their continued support, it would have taken me much longer to finish this project. I appreciate the support and guidance that I received from my committee members, Dr.
    [Show full text]