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Discrete Arginine Topologies Guide Escape of Miniature Proteins from Early Endosomes to the Cytoplasm Jacob S

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Discrete Arginine Topologies Guide Escape of Miniature Proteins from Early Endosomes to the Cytoplasm Jacob S Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 10-1-2012 Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm Jacob S. Appelbaum Yale University Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Appelbaum, Jacob S., "Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm" (2012). Yale Medicine Thesis Digital Library. 3362. http://elischolar.library.yale.edu/ymtdl/3362 This Open Access Dissertation is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm. A Dissertation Presented to the Faculty of the Graduate School of Yale University in Candidacy for the Degree of Doctor of Philosophy by Jacob S. Appelbaum Dissertation Director: Alanna Schepartz i October 2012 Abstract Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm. Jacob S. Appelbaum 2012 Polypeptides and peptide mimetics sample a wide chemical space with broad potential to modulate cellular function, but their application to cytoplasmic targets is limited be­ cause when added to cells their cytosolic concentration remains low. This limitation is due to a diffusion barrier (the plasma membrane) and absence of dedicated import ma­ chinery. Highly cationic peptides and proteins sometimes gain cytosolic access, but how they do so is not well understood. Using a small library of cationic miniature proteins, I probe the influence of positive charge number and orientation on the ability the minia­ ture protein to access the cytoplasm. Using a novel assay, I identify a cationic miniature protein, which we called 5.3, that carries a discrete arginine motif and efficiently reaches the cytoplasm. Database searches find that the precise motif identified (an arginine present in positions i, i + 4, i + 7, i + 10, and i + 11 of an a-helix) is not present in na­ ture, but that similar motifs are present in natural proteins that interact with cellular membranes. Finally, I examine the cellular pathway by which 5.3 reaches the cytoplasm. I find that this miniature protein enters the cell via a dynamin and cholesterol depen­ dent endocytic mechanism and is delivered to Rab5+ early endosomes. In contrast to the shiga-like toxins, and many non-enveloped viruses (which escape to the cytoplasm from the endoplasmic reticulum) as well as other peptides previously identified as 'cell penetrating', only 5.3 escapes from early endosomes. These findings should enable the future dissection of the precise molecular events underlying cytoplasmic access of pep­ tides and proteins, and may illuminate principles for the engineering of peptides and peptidomimetics that access cytoplasmic targets. Contents 1 Introduction 1 2 Use of Bipartite Tetracysteine Display to Probe the Association of Cross-Presented Peptides with the Major Histocompatability Complex 5 2.1 Background , 5 2.1.1 The spatial requirements of antigen presentation 6 2.1.2 Existing approaches to probe protein-protein interactions and their application to cross presentation 9 2.2 Results 10 2.2.1 A bipartite tetracysteine display strategy to selectively label peptide- MHC-i complexes 11 2.2.2 Expression analysis of generated MHC-i Variants 13 2.2.3 Recognition of tetracysteine motifs within oxidizing cellular envi­ ronments 18 2.2.4 Dicysteine HLA-A2 variants are not loaded with dicysteine-containing A2-binding peptides 20 2.3 Discussion 24 2.3.1 Future Directions I: Strategies for interrogating cross presentation . 26 2.3.2 Future Directions II: Targets of choice for bipartite tetracysteine dis­ play 27 ii 2-4 Experimental Methods 29 2.4.1 In silico mutagenesis and structural analysis of MHC-i and ReAsH- FLNCCPGCCMEP . 29 2.4.2 Generation of dicysteine containing HLA-A2-GFP variants 31 2.4.3 Immunofluorescence of HLA-A2-GFP and dicysteine containing vari­ ants 31 2.4.4 Flow cytometric analysis of surface-expression of HLA-A2-GFP and dicysteine containing variants 33 2.4.5 ReAsH staining of tetracysteine-tagged HLA-A2-GFP 34 2.4.6 Surface stabilization of dicysteine containing HLA-A2 variants by serum 35 2.4.7 Surface stabilization of dicysteine containing HLA-A2 variants by peptide • 35 3 Discrete Arginine Topologies Direct Cytosolic Access 36 3.1 Background 36 3.2 Results 38 3.2.1 Miniature Protein Design 38 3.2.2 Cell Uptake 39 3.2.3 Cationic miniature proteins do not cause plasma membrane dis­ ruption 45 3.2.4 Cationic Miniature Proteins Reach The Cytoplasm 47 3.2.5 Cell Penetrating Cationic Miniature Proteins Reach The Cytosol Intact 52 3.2.6 Cationic Miniature Protein 5.3 is a monomer 62 3.2.7 Natural Helices containing Arginine 68 3.2.7.1 An arginine-rich helix search 68 3.2.7.2 Functions of natural arginine rich helices 71 3.3 Discussion 76 iii 3.4 Experimental Methods 79 3.4.x Miniature protein synthesis and purification 79 3.4.1.x Fluorescein labeled miniature proteins 80 3.4.1.2 Rhodamine labeled miniature proteins and peptides. ... 82 3.4.1.3 SDEX labeled miniature proteins and peptides 82 3.4.2 Concentration Determination 83 3.4.3 Circular Dichroism 83 3.4.4 Activation of GR by dexamethasone labeled peptides and minia­ ture proteins 83 3.4.5 Image analysis using CellProfiler. 84 3.4.6 Examination of Membrane Integrity via Cell Impermeable Dyes. 85 3.4.7 Competition binding of dexamethasone labeled miniature proteins or peptides with Fluormone® for the glucocorticoid receptor. ... 85 3.4.8 Peptide Degradation 86 3.4.9 Resistance to Cathepsin Acid Proteases 86 3.4.10 Generation of Cytoplasmic Extracts using SLO 87 3.4.11 Analytical Ultracentrifugation 88 4 Cationic Miniature Proteins Reach The Cytoplasm By Escaping From Early En­ dosomes 93 4.1 Background 93 4.1.1 Endocytic mechanisms of cationic peptide internalization 94 4.1.2 Trafficking of endocytosed peptide 95 4.1.3 Reaching the cytoplasm 96 4.2 Results 97 4.2.1 Cationic miniature proteins traffic first into endocytic vesicles .... 97 4.2.2 Cytoplasmic access requires active endocytosis 103 4.2.3 Escape to the Cytoplasm from Early Endosomes 104 iv 4.2.4 Arginine topology affects protonation state transitions during en­ docytic acidification 113 4.3 Discussion 118 4.4 Experimental Methods 120 4.4.1 Cell Culture and Transfections 120 4.4.2 Colocalization of miniature proteins with Alexa-488-transferrin and Rab-GFP fusions 121 4.4.3 Effects of inhibitors on cell uptake 122 4.4.4 Effect of inhibitors on cytoplasmic access by dex-labeled miniature proteins or peptides 122 4.4.5 pGR-mCherry vector construction 123 4.4.6 Requirement of Rab5 activity for the ability of peptides to reach the cytoplasm 123 4.4.7 Statistical Analysis 124 Bibliography 126 v List of Figures 2.1 Pathways of Cross-presentation 8 2.2 A bipartite tetracysteine-based strategy, to detect peptide-MHC complexes 13 2.3 Microscopic analysis of the subcellular distribution of GFP-tagged dicys­ teine containing HLA-A2 variants 15 2.4 Surface expression of dicysteine containing HLA-A2 variants as assessed by flow cytometry 17 2.5 Detection of tetracysteine motifs within oxidizing environments 19 2.6 Surface stabilization of dicysteine containing HLA-A2 variants on cells lacking TAP 21 3.1 Description of miniature protein structures and variation in arginine display 40 3.2 Circular dichroism (CD) of miniature proteins used in this study. 42 3.3 Cell uptake of cationic miniature proteins 43 3.4 Absence of membrane permeabilization after treatment with Dex-labeled cationic miniature proteins 46 3.5 Dose dependent increase in translocation ratio following exposure to dex­ amethasone 49 3.6 Translocation of GR-GFP after treatment with dexamethasone and dex- labeled miniature proteins 53 3.7 Competition binding of dexamethasone labeled cationic miniature pro­ teins or peptides with GR 54 vi 3.8 Absence of degradation of cationic miniature proteins 55 3.9 Absence of intracellular degradation of cationic miniature proteins 56 3.10 Confirmation that SLO induces cell permeabilization 59 3.11 Recovery of cationic miniature protein 5.3, unmodified, from the cytoplasm 61 3.12 Direct observation that the 5.3 motif increases the cytoplasmic presence of miniature proteins 63 3.13 Analysis of aPP and its dexamethasone labeled variant by analytical ultra- centrifugation 65 3.14 Association states of aPP and aPPDex as they vary with concentration ... 69 3.15 Cationic miniature protein 5.3 and its dexamethasone labeled variant are monomers 69 3.16 Schema for the identification of arginine-rich helices from the PDB 72 3.17 Natural proteins containing arginine-rich helices 74 3.18 HPLC analysis of selected cell permeable miniature proteins 81 3.19 HPLC analysis of labeled peptides used in this study 91 3.20 Mass spectral data obtained for miniature proteins and peptides in this work 92 4.1 Colocalization of Tf488 with rhodamine labeled miniature proteins 99 4.2 Colocalization of rhodamine and fluorescein labeled cationic miniature proteins 101 4.3 Uptake of cationic miniature proteins is inhibited by treatments the block endocytosis 102 4.4 Inhibition of endocytosis blocks cytoplasmic access 105 4.5 Miniature protein 5.3s enters via endocytosis into Rab5+ vesicles 108 4.6 Cell permeable cationic miniature proteins are not found within the Golgi 109 4.7 Cationic miniature proteins share an overlapping intracellular distribution 110 4.8 Cationic miniature protein 5.3 escapes from early endosomes 112 vii Arginine interactions reveals perturb side-chain pKa's, stabilizing depro- tonated carboxyl groups 116 viii Acknowledgements Much of the work described in chapters 3 and 4 was recently submitted as a manuscript for publication.
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