DOCSLIB.ORG

ABSTRACT Title of Thesis: INVESTIGATION BY

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ABSTRACT Title of Thesis: INVESTIGATION BY ABSTRACT Title of Thesis: INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS Katherine R. Adams, Master of Science, 2016 Thesis Directed By: Catherine Fenselau, Professor, Department of Chemistry and Biochemistry Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes. INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS by Katherine R. Adams Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2016 Advisory Committee: Professor Catherine Fenselau, Chair Professor Neil Blough Professor Nicole LaRonde © Copyright by Katherine R. Adams 2016 Dedication This work is dedicated to my parents, David and Mary Lou Adams, who have always supported me in all walks of life and taught me the value of hard work. To my older brother Matthew who taught me all of life’s important lessons. And to my boyfriend and best friend Adam who was always available for a pep talk and food. ii Acknowledgements First and foremost I would like to acknowledge my advisor Dr. Catherine Fenselau. It has been such an honor to work in this research group these past two years and learn as much as I could. Thank you for your guidance and invaluable wisdom that I will carry with me throughout my career. I would like to acknowledge my collaborators who have made this project possible. I would like to thank Dr. Suzanne Ostrand-Rosenberg and Virginia Clements for providing the MDSC exosomes and for help with understanding the cell biology. I would also like to thank Dr. Yan Wang for help and guidance using the orbitrap Fusion Lumos, an amazing and incredibly expensive instrument that has been a highlight of my graduate school work. I would like to thank Dr. Nathan Edwards for always being available to answer bioinformatics questions. I would also like to thank my committee members Dr. Neil Blough and Dr. Nicole LaRonde. I would like to thank all past and current members of the Fenselau group including Lucia Geis Asteggiante, Sitara Chauhan, Yeji Kim, Mrs. Sara Moran, Dr. Dapeng Chen, Dr. Amanda Lee, and Dr. Meghan Burke. I would also like to thank the amazing group of graduate students I have met while at Maryland; my gal pals Leila Duman, Teodora Kljaic, Sarah Robinson, and Emily Sahadeo, my crazy fun roommates Noah Masika, Kyle Oliver, and Vincent Wu, and an extra special thank you to my soul friend Samantha Nowak who I will never stop being thankful for. Lastly I would like to thank my family for their unconditional love and support. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables ................................................................................................................ v List of Figures .............................................................................................................. vi List of Abbreviations ................................................................................................. viii Chapter 1: Introduction ................................................................................................. 1 1.1 Proteomics........................................................................................................... 1 1.1.1 Overview ...................................................................................................... 1 1.1.2 Orbitrap Fusion Lumos Tribrid .................................................................... 5 1.2 Biological question ............................................................................................. 7 1.2.1 Exosome biogenesis and function ................................................................ 7 1.2.2 Ubiquitin as a post-translational modification ............................................. 8 1.2.3 The role of ubiquitin in exosome formation .............................................. 11 1.3 Research objectives and significance ................................................................ 12 Chapter 2: Identification of proteins in the exosome lysate ....................................... 14 2.1 Introduction ....................................................................................................... 14 2.2 Materials and methods ...................................................................................... 16 2.2.1 Materials .................................................................................................... 16 2.2.2 Biological sample preparation ................................................................... 16 2.2.3 Protein analysis .......................................................................................... 16 2.2.4 LC-MS/MS and bioinformatics ................................................................. 17 2.3 Results and discussion ...................................................................................... 19 2.4 Conclusions ....................................................................................................... 27 Chapter 3: The Ubiquitome ........................................................................................ 29 3.1 Introduction ....................................................................................................... 29 3.2 Materials and methods ...................................................................................... 32 3.2.1 Materials .................................................................................................... 32 3.2.2 Biological sample preparation and protein analysis .................................. 32 3.2.3 Immunoprecipitations ................................................................................ 33 3.2.4 Western blotting ......................................................................................... 34 3.3 Results and discussion ...................................................................................... 34 3.3.1 Confirmation of ubiquitinated proteins by immunoprecipitation .............. 34 3.3.2 Bottom-up identification of ubiquitinated proteins.................................... 36 3.4 Conclusions ....................................................................................................... 42 Appendices .................................................................................................................. 45 Bibliography ............................................................................................................... 86 iv List of Tables Table 2.1 – Some biologically active proteins identified in the exosome lysate. Table 2.2 – Proteasome components identified in the exosome lysate. Table 2.3 – Ubiquitination pathway enzymes identified in the exosome lysate. v List of Figures Figure 1.1. Diagram of the fragmentation and ion types that can occur along the peptide backbone. Figure 1.2. Schematic of the orbitrap Fusion Lumos Tribrid mass spectrometer. (http://planetorbitrap.com/) Figure 1.3. Illustration of the formation of multi-vesicular bodies and intraluminal vesicles, and their pathway to degradation in the lysosome or release as exosomes. Figure 1.4. An overview of the components involved in the ubiquitin system in mammalian cells. Figure 2.1. A flowchart summarizing sample preparation and this bottom-up workflow. Figure 2.2a-b. GO annotations of the (a) molecular functions and (b) cellular components for the whole exosome lysate. Figure 2.3a-b. (a) Isoelectric point, pI, distribution of the proteins identified in the exosome lysate and (b) a comparison of the isoelectric point distributions of the exosome lysate and mouse proteome (available from http://isoelectricpointdb.org/index.html). Figure 2.4. MW distributions of the proteins identified in the MDSC exosomes; inset shows the 0 - 350,000 Da range. Figure 3.1. A bottom-up proteomic workflow for ubiquitinated proteins utilizing digestion by trypsin and activation by CID. (Adapted from references 11 and 39). vi Figure 3.2a-f. Western blots probing with anti-ubiquitin antibody #3933 of (a) exosome lysate, (b) eluate of #3933 immunoprecipitation, (c) non-bound fraction from #3933 immunoprecipitation, (d) negative control of #3933 immunoprecipitation, (e) eluate
Load more

Recommended publications
  • Pathway-Specific Dopaminergic Deficits in a Mouse Model of Angelman Syndrome
    Pathway-specific dopaminergic deficits in a mouse model of Angelman syndrome Thorfinn T. Riday, … , Benjamin D. Philpot, C.J. Malanga J Clin Invest. 2012;122(12):4544-4554. https://doi.org/10.1172/JCI61888. Research Article Neuroscience Angelman syndrome (AS) is a neurodevelopmental disorder caused by maternal deletions or mutations of the ubiquitin ligase E3A (UBE3A) allele and characterized by minimal verbal communication, seizures, and disorders of voluntary movement. Previous studies have suggested that abnormal dopamine neurotransmission may underlie some of these deficits, but no effective treatment currently exists for the core features of AS. A clinical trial of levodopa (l-DOPA) in AS is ongoing, although the underlying rationale for this treatment strategy has not yet been thoroughly examined in preclinical models. We found that AS model mice lacking maternal Ube3a (Ube3am–/p+ mice) exhibit behavioral deficits that correlated with abnormal dopamine signaling. These deficits were not due to loss of dopaminergic neurons or impaired dopamine synthesis. Unexpectedly, Ube3am–/p+ mice exhibited increased dopamine release in the mesolimbic pathway while also exhibiting a decrease in dopamine release in the nigrostriatal pathway, as measured with fast-scan cyclic voltammetry. These findings demonstrate the complex effects of UBE3A loss on dopamine signaling in subcortical motor pathways that may inform ongoing clinical trials of l-DOPA therapy in patients with AS. Find the latest version: https://jci.me/61888/pdf Research article Pathway-specific dopaminergic deficits in a mouse model of Angelman syndrome Thorfinn T. Riday,1,2 Elyse C. Dankoski,1 Michael C. Krouse,3 Eric W. Fish,3 Paul L.
    [Show full text]
  • Supplementary Information Method CLEAR-CLIP. Mouse Keratinocytes
    Supplementary Information Method CLEAR-CLIP. Mouse keratinocytes of the designated genotype were maintained in E-low calcium medium. Inducible cells were treated with 3 ug/ml final concentration doxycycline for 24 hours before performing CLEAR-CLIP. One 15cm dish of confluent cells was used per sample. Cells were washed once with cold PBS. 10mls of cold PBS was then added and cells were irradiated with 300mJ/cm2 UVC (254nM wavelength). Cells were then scraped from the plates in cold PBS and pelleted by centrifugation at 1,000g for 2 minutes. Pellets were frozen at -80oC until needed. Cells were then lysed on ice with occasional vortexing in 1ml of lysis buffer (50mM Tris-HCl pH 7.4, 100mM NaCl, 1mM MgCl2, 0.1 mM CaCl2, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) containing 1X protease inhibitors (Roche #88665) and RNaseOUT (Invitrogen #10777019) at 4ul/ml final concentration. Next, TurboDNase (Invitrogen #AM2238, 10U), RNase A (0.13ug) and RNase T1 (0.13U) were added and samples were incubated at 37oC for 5 minutes with occasional mixing. Samples were immediately placed on ice and then centrifuged at 16,160g at 4oC for 20 minutes to clear lysate. 25ul of Protein-G Dynabeads (Invitrogen #10004D) were used per IP. Dynabeads were pre-washed with lysis buffer and pre- incubated with 3ul of Wako Anti-Mouse-Ago2 (2D4) antibody. The dynabead/antibody mixture was added to the lysate and rocked for 2 hours at 4oC. All steps after the IP were done on bead until samples were loaded into the polyacrylamide gel.
    [Show full text]
  • Transcriptomic Profile Induced in Bone Marrow Mesenchymal Stromal Cells After Interaction with Multiple Myeloma Cells
    www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 18 Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease Antonio Garcia-Gomez1,2,3, Javier De Las Rivas1, Enrique M. Ocio1,2, Elena Díaz- Rodríguez1, Juan C. Montero1, Montserrat Martín1,3, Juan F. Blanco2, Fermín M. Sanchez-Guijo2,3, Atanasio Pandiella1,2, Jesús F. San Miguel1,2,3 and Mercedes Garayoa1,2,3 1 Centro de Investigación del Cáncer, IBMCC (Universidad de Salamanca-CSIC), Salamanca, Spain 2 Hospital Universitario de Salamanca-IBSAL, Salamanca, Spain 3 Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain Correspondence to: Mercedes Garayoa, email: [email protected] Keywords: multiple myeloma, bone marrow mesenchymal stromal cells, tumor-stroma interactions, gene expression profiling, co- culture techniques, myeloma bone disease Received: January 6, 2014 Accepted: June 2, 2014 Published: June 4, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co- cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross- talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition.
    [Show full text]
  • The Autism Protein Ube3a/E6AP Remodels Neuronal Dendritic Arborization Via Caspase-Dependent Microtubule Destabilization
    The Journal of Neuroscience, January 10, 2018 • 38(2):363–378 • 363 Neurobiology of Disease The Autism Protein Ube3A/E6AP Remodels Neuronal Dendritic Arborization via Caspase-Dependent Microtubule Destabilization Natasha Khatri,1,2 James P. Gilbert,1 Yuda Huo,1 Roozhin Sharaflari,1 Michael Nee,1 Hui Qiao,1 and XHeng-Ye Man1,2 1Department of Biology, Boston University, Boston, Massachusetts 02215, and 2Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118 UBE3A gene copy number variation and the resulting overexpression of the protein E6AP is directly linked to autism spectrum disorders (ASDs).However,theunderlyingcellularandmolecularneurobiologyremainslessclear.HerewereporttheroleofASD-relatedincreased dosage of Ube3A/E6AP in dendritic arborization during brain development. We show that increased E6AP expression in primary cul- tured neurons leads to a reduction in dendritic branch number and length. The E6AP-dependent remodeling of dendritic arborization results from retraction of dendrites by thinning and fragmentation at the tips of dendrite branches, leading to shortening or removal of dendrites. This remodeling effect is mediated by the ubiquitination and degradation of XIAP (X-linked inhibitors of aptosis protein) by E6AP, which leads to activation of caspase-3 and cleavage of microtubules. In vivo, male and female Ube3A 2X ASD mice show decreased XIAP levels, increased caspase-3 activation, and elevated levels of tubulin cleavage. Consistently, dendritic branching and spine density are reduced in cortical neurons of Ube3A 2X ASD mice. In revealing an important role for Ube3A/E6AP in ASD-related developmental alteration in dendritic arborization and synapse formation, our findings provide new insights into the pathogenesis of Ube3A/E6AP- dependent ASD.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Supplementary Table 9. Functional Annotation Clustering Results for the Union (GS3) of the Top Genes from the SNP-Level and Gene-Based Analyses (See ST4)
    Supplementary Table 9. Functional Annotation Clustering Results for the union (GS3) of the top genes from the SNP-level and Gene-based analyses (see ST4) Column Header Key Annotation Cluster Name of cluster, sorted by descending Enrichment score Enrichment Score EASE enrichment score for functional annotation cluster Category Pathway Database Term Pathway name/Identifier Count Number of genes in the submitted list in the specified term % Percentage of identified genes in the submitted list associated with the specified term PValue Significance level associated with the EASE enrichment score for the term Genes List of genes present in the term List Total Number of genes from the submitted list present in the category Pop Hits Number of genes involved in the specified term (category-specific) Pop Total Number of genes in the human genome background (category-specific) Fold Enrichment Ratio of the proportion of count to list total and population hits to population total Bonferroni Bonferroni adjustment of p-value Benjamini Benjamini adjustment of p-value FDR False Discovery Rate of p-value (percent form) Annotation Cluster 1 Enrichment Score: 3.8978262119731335 Category Term Count % PValue Genes List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDR GOTERM_CC_DIRECT GO:0005886~plasma membrane 383 24.33290978 5.74E-05 SLC9A9, XRCC5, HRAS, CHMP3, ATP1B2, EFNA1, OSMR, SLC9A3, EFNA3, UTRN, SYT6, ZNRF2, APP, AT1425 4121 18224 1.18857065 0.038655922 0.038655922 0.086284383 UP_KEYWORDS Membrane 626 39.77128335 1.53E-04 SLC9A9, HRAS,
    [Show full text]
  • Targeting the Ubiquitin System in Glioblastoma', Frontiers in Oncology
    Citation for published version: Licchesi, J 2020, 'Targeting the Ubiquitin System in Glioblastoma', Frontiers in Oncology. https://doi.org/10.3389/fonc.2020.574011 DOI: 10.3389/fonc.2020.574011 Publication date: 2020 Document Version Publisher's PDF, also known as Version of record Link to publication University of Bath Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Sep. 2021 REVIEW published: 25 November 2020 doi: 10.3389/fonc.2020.574011 Targeting the Ubiquitin System in Glioblastoma Nico Scholz 1, Kathreena M. Kurian 2, Florian A. Siebzehnrubl 3 and Julien D. F. Licchesi 1* 1 Department of Biology & Biochemistry, University of Bath, Bath, United Kingdom, 2 Brain Tumour Research Group, Institute of Clinical Neurosciences, University of Bristol, Bristol, United Kingdom, 3 Cardiff University School of Biosciences, European Cancer Stem Cell Research Institute, Cardiff, United Kingdom Glioblastoma is the most common primary brain tumor in adults with poor overall outcome and 5-year survival of less than 5%. Treatment has not changed much in the last decade or so, with surgical resection and radio/chemotherapy being the main options.
    [Show full text]
  • Progression of Pathology in PINK1-Deficient Mouse Brain From
    Torres-Odio et al. Journal of Neuroinflammation (2017) 14:154 DOI 10.1186/s12974-017-0928-0 RESEARCH Open Access Progression of pathology in PINK1-deficient mouse brain from splicing via ubiquitination, ER stress, and mitophagy changes to neuroinflammation Sylvia Torres-Odio1†, Jana Key1†, Hans-Hermann Hoepken1, Júlia Canet-Pons1, Lucie Valek2, Bastian Roller3, Michael Walter4, Blas Morales-Gordo5, David Meierhofer6, Patrick N. Harter3, Michel Mittelbronn3,7,8,9,10, Irmgard Tegeder2, Suzana Gispert1 and Georg Auburger1* Abstract Background: PINK1 deficiency causes the autosomal recessive PARK6 variant of Parkinson’s disease. PINK1 activates ubiquitin by phosphorylation and cooperates with the downstream ubiquitin ligase PARKIN, to exert quality control and control autophagic degradation of mitochondria and of misfolded proteins in all cell types. Methods: Global transcriptome profiling of mouse brain and neuron cultures were assessed in protein-protein interaction diagrams and by pathway enrichment algorithms. Validation by quantitative reverse transcriptase polymerase chain reaction and immunoblots was performed, including human neuroblastoma cells and patient primary skin fibroblasts. Results: In a first approach, we documented Pink1-deleted mice across the lifespan regarding brain mRNAs. The expression changes were always subtle, consistently affecting “intracellular membrane-bounded organelles”.Significant anomalies involved about 250 factors at age 6 weeks, 1300 at 6 months, and more than 3500 at age 18 months in the cerebellar tissue, including Srsf10, Ube3a, Mapk8, Creb3,andNfkbia. Initially, mildly significant pathway enrichment for the spliceosome was apparent. Later, highly significant networks of ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing occurred. Finally, an enrichment of neuroinflammation factors appeared, together with profiles of bacterial invasion and MAPK signaling changes—while mitophagy had minor significance.
    [Show full text]
  • Supplementary Material Table of Contents
    Supplementary M aterial Table of Contents 1 - Online S u pplementary Methods ………………………………………………...… . …2 1.1 Study population……………………………………………………………..2 1.2 Quality control and principal component analysis …………………………..2 1.3 Statistical analyses………………………………………… ………………...3 1.3.1 Disease - specific association testing ……………………………… ..3 1.3.2 Cross - phenotype meta - analysis …………………………………… .3 1.3.3 Model search ……………………………………………………… .4 1.3.4 Novelty of the variants …………………………………………… ..4 1.3.5 Functional Enrichment Analy sis ………………………………… ...4 1.3.6 Drug Target Enrichment Analysis ………………………………… 5 2 - Supplementary Figures………………………………………...………………… . …. 7 3 - Members of the Myositis Genetics Consortium (MYOGEN) ……………………. ..16 4 - Members of the Scleroderma Genetics Consortium ………………… ……………...17 5 - Supplementary References………………………………………………………… . .18 6 - Supplementary Tables………………………………………………………… . ……22 1 Online supplementary m ethods Study population This study was conducted using 12,132 affected subjects and 23 ,260 controls of European des cent population and all of them have been included in previously published GWAS as summarized in Table S1. [1 - 6] Briefly, a total of 3,255 SLE cases and 9,562 ancestry matched controls were included from six countrie s across Europe and North America (Spain, Germany, Netherlands, Italy, UK, and USA). All of the included patients were diagnosed based on the standard American College of Rheumatology (ACR) classification criteria. [7] Previously described GWAS data from 2,363 SSc cases and 5,181 ancestry
    [Show full text]
  • The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-To-ER Retrograde Transport
    Supplementary Materials The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport Bustamante HA., et al. Figure S1. siRNA sequences directed against human PSMD14 used for Validation Stage. Figure S2. Primer pairs sequences used for RT-qPCR. Figure S3. The PSMD14 DUB inhibitor CZM increases the Golgi apparatus area. Immunofluorescence microscopy analysis of the Golgi area in parental H4 cells treated for 4 h either with the vehicle (DMSO; Control) or CZM. The Golgi marker GM130 was used to determine the region of interest in each condition. Statistical significance was determined by Student's t-test. Bars represent the mean ± SEM (n =43 cells). ***P <0.001. Figure S4. CZM causes the accumulation of KDELR1-GFP at the Golgi apparatus. HeLa cells expressing KDELR1-GFP were either left untreated or treated with CZM for 30, 60 or 90 min. Cells were fixed and representative confocal images were acquired. Figure S5. Effect of CZM on proteasome activity. Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM or MG132, for 90 min. Protein extracts were used to measure in vitro the Chymotrypsin-like peptidase activity of the proteasome. The enzymatic activity was quantified according to the cleavage of the fluorogenic substrate Suc-LLVY-AMC to AMC, and normalized to that of control cells. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). **P <0.01; n.s., not significant. Figure S6. Effect of CZM and MG132 on basal macroautophagy. (A) Immunofluorescence microscopy analysis of the subcellular localization of LC3 in parental H4 cells treated with either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h.
    [Show full text]
  • Aggresomal Sequestration and STUB1-Mediated Ubiquitylation During Mammalian Proteaphagy of Inhibited Proteasomes
    Aggresomal sequestration and STUB1-mediated ubiquitylation during mammalian proteaphagy of inhibited proteasomes Won Hoon Choia,b, Yejin Yuna,b, Seoyoung Parka,c, Jun Hyoung Jeona,b, Jeeyoung Leea,b, Jung Hoon Leea,c, Su-A Yangd, Nak-Kyoon Kime, Chan Hoon Jungb, Yong Tae Kwonb, Dohyun Hanf, Sang Min Lime, and Min Jae Leea,b,c,1 aDepartment of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 03080 Seoul, Korea; bDepartment of Biomedical Sciences, Seoul National University Graduate School, 03080 Seoul, Korea; cNeuroscience Research Institute, Seoul National University College of Medicine, 03080 Seoul, Korea; dScience Division, Tomocube, 34109 Daejeon, Korea; eConvergence Research Center for Diagnosis, Korea Institute of Science and Technology, 02792 Seoul, Korea; and fProteomics Core Facility, Biomedical Research Institute, Seoul National University Hospital, 03080 Seoul, Korea Edited by Richard D. Vierstra, Washington University in St. Louis, St. Louis, MO, and approved July 1, 2020 (received for review November 18, 2019) The 26S proteasome, a self-compartmentalized protease complex, additional LC3-interacting region; the target cargoes can be plays a crucial role in protein quality control. Multiple levels of docked onto phosphatidylethanolamine-modified LC3 (LC3-II) regulatory systems modulate proteasomal activity for substrate on the expanding phagophore membrane, enveloped by an hydrolysis. However, the destruction mechanism of mammalian autophagosome, and eventually degraded in the autolysosomes. proteasomes is poorly understood. We found that inhibited pro- Notably, the enzymatic cascade attaching the lipid moiety at the teasomes are sequestered into the insoluble aggresome via C-terminal glycine of the cleaved LC3 protein in autophagy re- HDAC6- and dynein-mediated transport.
    [Show full text]
  • The Urea Cycle and the De Novo Human Mutome I
    The urea cycle and the de novo human mutome Ana Sofia Mendes Oliveira Mestrado em Genética Forense Departamento de Biologia 2017 Orientador Luísa Azevedo, PhD, Faculdade de Ciências da Universidade do Porto (FCUP), Instituo de Patologia e Imunologia Molecular da Universidade do Porto (Ipatimup), Instituto de Investigação e Inovação em Saúde (i3S). Coorientador Manuela Oliveira, PhD, Faculdade de Ciências da Universidade do Porto (FCUP), Instituo de Patologia e Imunologia Molecular da Universidade do Porto (Ipatimup), Instituto de Investigação e Inovação em Saúde (i3S). All corrections determined by the jury, and only those, were incorporated. The President of the Jury, Porto, ______/______/_________ FCUP The urea cycle and the de novo human mutome i Acknowledgments Once completed a new step of my life is time to thank all the people who inspired me, who accompanied me and who contributed to the success of this step. First, I would like to thank my supervisor Luísa Azevedo for having accepted the guidance of this project. A huge thank you for guidance, for the constant availability and for the support and understanding. I would also like to thank my co-supervisor, Manuela Oliveira, for contributing to the improvement of my project by giving new ideas and suggestions. I am also indebted to Professor, António Amorim, for having contributed to my education. To my dear parents, Paula and David, for their knowledge, for their love and affection, understanding and dedication; for all the support that helped me to achieve my goals. I am very grateful to you both for the person I am today.
    [Show full text]