DOCSLIB.ORG

Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation Barbara Bertocci, Damiana Lecoeuche, Delphine Sterlin, Julius Kühn, Baptiste Gaillard, Annie De Smet, Frederique This information is current as Lembo, Christine Bole-Feysot, Nicolas Cagnard, Tatiana of September 28, 2021. Fadeev, Auriel Dahan, Jean-Claude Weill and Claude-Agnès Reynaud J Immunol 2017; 199:2408-2420; Prepublished online 14 August 2017; Downloaded from doi: 10.4049/jimmunol.1700708 http://www.jimmunol.org/content/199/7/2408 Supplementary http://www.jimmunol.org/content/suppl/2017/08/11/jimmunol.170070 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 60 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/199/7/2408.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation Barbara Bertocci,* Damiana Lecoeuche,* Delphine Sterlin,*,1 Julius Kuhn,€ † Baptiste Gaillard,*,2 Annie De Smet,* Frederique Lembo,‡ Christine Bole-Feysot,x Nicolas Cagnard,{ Tatiana Fadeev,* Auriel Dahan,* Jean-Claude Weill,* and Claude-Agne`s Reynaud* Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have Downloaded from passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt’s lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that http://www.jimmunol.org/ KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process. The Journal of Immunology, 2017, 199: 2408–2420. cell generation follows a stepwise process of differenti- guish T1 (CD93+CD232) from T2 B cells (CD93+CD23+) (6–8). It ation, first in the bone marrow and later in the periphery, has been observed that immature B cells can also differentiate into B where fully mature B cells reside. After a productive T1-like and T2-like B cells within the bone marrow (9, 10). T2 rearrangement and expression of a functional BCR at the surface, B cells, which emerge in spleen with a parallel but slightly B cells are still at an immature stage. Cross-linking of the BCR at delayed timing, could thus originate from both in situ T1 cell by guest on September 28, 2021 this stage induces cell death, a negative selection process purging differentiation and migration of T2-like cells from bone marrow the B cell repertoire from autoreactive specificities (1, 2). (11, 12). Interaction between the B cell–activating cytokine BAFF Immature B cells that survive past this checkpoint migrate to the and its specific receptor (BAFF-R) on B cells appears to play spleen and mature into follicular (FO) and marginal zone (MZ) different roles in this process, depending on the B cell differen- B cells (3). This maturation occurs in successive steps and implies tiation stage and the lymphoid organ concerned (13–19). Whereas the formation of two subsets of transitional B cells, transitional the immature to T1-like B cell progression in bone marrow seems type 1 (T1) and transitional type 2 (T2) (4), which have been to occur in a BAFF-independent mode, this is not the case in the proposed to be located in different areas of the spleen, the peri- spleen, where T1 cells require BAFF signals for differentiation arteriolar lymphoid sheath and the B cell follicles, respectively and/or survival, as shown in mixed bone marrow chimeras with (5). Several surface markers have been used to characterize T1 and BAFF-R–deficient and BAFF-R–competent cells (20). Conversely, T2 B cells, notably expression of CD93 and CD23, which distin- the differentiation of both splenic and bone marrow T1 into T2 *E´ quipe De´veloppement du Syste´me Immunitaire, Institut Necker-Enfant Malades, This work was supported by the Fondation Princesse Grace (to Team De´veloppement INSERM U1151-CNRS UMR8253, Faculte´ de Me´decine Paris Decartes, Universite´ du Syste`me Immunitaire) and the Ligue contre le Cancer (E´ quipe Labellise´e). Paris Descartes, Sorbone Paris Cite´, 75993 Paris Cedex 14, France; †Institute of The microarray data presented in this article have been submitted to the ArrayEx- Cellular and Molecular Immunology, Georg-August-University Medicine Go¨ttingen, press database (https://www.ebi.ac.uk/arrayexpress/browse.html) under accession 37073 Go¨ttingen, Germany; ‡Centre de Recherche en Cance´rologie de Marseille, x number E-MTAB-5928. INSERM U1068-CNRS UMR7258, 13273 Marseille Cedex 09, France; Plateforme de Ge´nomique, Imagine Institut des Maladies Ge´ne´tiques-Structure Fe´de´rative de Address correspondence and reprint requests to Dr. Barbara Bertocci, E´ quipe De´vel- Recherche Necker, INSERM 1163 and INSERM US24/CNRS UMS3633, 75015 oppement du Syste´me Immunitaire, Institut Necker-Enfant Malades, INSERM Paris, France; and {Plateforme de Bioinformatique, Universite´ Paris Descartes- U1151-CNRS UMR8253, Faculte´ de Me´decine Paris Decartes, Universite´ Paris Des- Structure Fe´de´rative de Recherche Necker, INSERM US24/CNRS UMS3633, cartes, Sorbone Paris Cite´, 14 rue Maria Helena Vieira Da Silva, Paris Cedex 14, 75993 Paris Cedex 14, France 75993 France. E-mail address: [email protected] 1Current address: Sorbonne Universite´s, Universite´ Pierre et Marie Curie/Universite´ The online version of this article contains supplemental material. Paris 6, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses Paris Abbreviations used in this article: AID, activation-induced cytidine deaminase; APC/ UMRS 1135, Assistance Publique – Hoˆpitaux de Paris, Groupement Hospitalier C, anaphase-promoting complex/cyclosome; CLL, chronic lymphocytic leukemia; Pitie´-Salpeˆtrie`re, De´partement d’Immunologie, Paris, France. Cul3, Cullin 3; FO, follicular; GC, germinal center; GSEA, gene set enrichment 2Current address: Service d’He´matologie, Hopital Robert Debre, Centre Hospitalier analysis; KO, knockout; MZ, marginal zone; PC, plasma cell; PLA, proximity liga- Universitaire de Reims, Reims, France. tion assay; T1, transitional type 1; T2, transitional type 2; wt, wild type. ORCIDs: 0000-0002-5532-9431 (T.F.); 0000-0002-4519-4473 (C.-A.R.). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Received for publication May 12, 2017. Accepted for publication July 17, 2017. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700708 The Journal of Immunology 2409 B cells is strongly dependent on BAFF signaling, because B cell Flow cytometry maturation in BAFF- and BAFF-R–deficient mice does not pro- Tissue samples were collected from the appropriate mice as described ceed beyond the T1 stage (13, 15, 19). in the text. After RBC lysis, cells were stained with a combination of The KLHL gene family encodes 42 different proteins, defined fluorophore-conjugated Abs (Supplemental Table I). Surface markers as being composed of an N-terminal BTB-POZ domain followed were detected and analyzed using a FACSCanto II flow cytometry ap- by a BTB and C-terminal kelch (BACK) domain and four to six paratus and the BD FACSDiva software (BD Biosciences). tandem Kelch motifs (21). The BTB domain, named by its ho- Cell culture mology with the Drosophila Bric-a-brac, Tramtrac, and Broad complex, is involved in protein–protein interaction and protein Splenic B cells, after purification (B cell isolation kit, MACS separation), were cultured in complete RPMI 1640 medium supplemented with 10% self-oligomerization (22). The BACK domain is very conserved, HyClone FetalClone I serum (Thermo Scientific), 100 U/ml penicillin and but its role is not unequivocally identified (23). The Kelch motif, 100 U/ml streptomycin, 1 mM sodium pyruvate, MEM nonessential which was discovered as a six-repeat element in the Drosophila amino acids (13), 25 mM HEPES (Invitrogen), 0.5 mM 2-ME (Sigma), kelch protein, is a segment of 44–56 aa that adopts a b-propeller and the indicated activating factors. All reagents, including anti-IgM (Jackson ImmunoResearch), LPS, CpG (Sigma), and recombinant BAFF structure and contains multiple potential protein contact sites protein (RD Biosystem), were prepared as per manufacturer’s instructions (24).
Load more

Recommended publications
  • Kelch Proteins: Emerging Roles in Skeletal Muscle Development and Diseases
    Kelch proteins: emerging roles in skeletal muscle development and diseases The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Gupta, Vandana A., and Alan H Beggs. 2014. “Kelch proteins: emerging roles in skeletal muscle development and diseases.” Skeletal Muscle 4 (1): 11. doi:10.1186/2044-5040-4-11. http:// dx.doi.org/10.1186/2044-5040-4-11. Published Version doi:10.1186/2044-5040-4-11 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12406733 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Gupta and Beggs Skeletal Muscle 2014, 4:11 http://www.skeletalmusclejournal.com/content/4/1/11 REVIEW Open Access Kelch proteins: emerging roles in skeletal muscle development and diseases Vandana A Gupta and Alan H Beggs* Abstract Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions.
    [Show full text]
  • The Genome of Swinepox Virus
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Virology Papers Virology, Nebraska Center for 1-1-2002 The Genome of Swinepox Virus C. L. Afonso Agricultural Research Service, U.S. Department of Agriculture E. R. Tulman Agricultural Research Service, U.S. Department of Agriculture Z. Lu Agricultural Research Service, U.S. Department of Agriculture L. Zsak Agricultural Research Service, U.S. Department of Agriculture Fernando A. Osorio University of Nebraska-Lincoln, [email protected] See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/virologypub Part of the Virology Commons Afonso, C. L.; Tulman, E. R.; Lu, Z.; Zsak, L.; Osorio, Fernando A.; Balinsky, C.; Kutish, G. F.; and Rock, D. L., "The Genome of Swinepox Virus" (2002). Virology Papers. 71. https://digitalcommons.unl.edu/virologypub/71 This Article is brought to you for free and open access by the Virology, Nebraska Center for at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Virology Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors C. L. Afonso, E. R. Tulman, Z. Lu, L. Zsak, Fernando A. Osorio, C. Balinsky, G. F. Kutish, and D. L. Rock This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ virologypub/71 JOURNAL OF VIROLOGY, Jan. 2002, p. 783–790 Vol. 76, No. 2 0022-538X/02/$04.00ϩ0 DOI: 10.1128/JVI.76.2.783–790.2002 The Genome of Swinepox Virus C. L. Afonso,1*E.R.Tulman,1 Z. Lu,1 L. Zsak,1 F.
    [Show full text]
  • Structural–Functional Interactions of NS1-BP Protein with the Splicing and Mrna Export Machineries for Viral and Host Gene Expression
    Structural–functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression Ke Zhanga, Guijun Shangb, Abhilash Padavannilb, Juan Wanga, Ramanavelan Sakthivela, Xiang Chenc,d, Min Kime, Matthew G. Thompsonf, Adolfo García-Sastreg,h,i, Kristen W. Lynchf, Zhijian J. Chenc,d, Yuh Min Chookb,1, and Beatriz M. A. Fontouraa,1 aDepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; cDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; dHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; eDepartment of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75390; fDepartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; gDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; hGlobal Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and iDivision of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved November 13, 2018 (received for review October 23, 2018) The influenza virulence factor NS1 protein interacts with the Viral mRNA splicing requires the cellular spliceosome. Most cellular NS1-BP protein to promote splicing and nuclear export of of cellular splicing occurs in the nucleoplasm, where splicing the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix factors are recruited to nascent transcripts through interaction protein and is alternatively spliced into the M2 mRNA, which is with polymerase II (Pol II).
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 6,989,232 B2 Burgess Et Al
    USOO6989232B2 (12) United States Patent (10) Patent No.: US 6,989,232 B2 Burgess et al. (45) Date of Patent: Jan. 24, 2006 (54) PROTEINS AND NUCLEIC ACIDS Adams et al, Nature 377 (Suppl), 3 (1995).* ENCODING SAME Nakayama et al, Genomics 51(1), 27(1998).* Mahairas et al. Accession No. B45150 (Oct. 21, 1997).* (75) Inventors: Catherine E. Burgess, Wethersfield, Wallace et al, Methods Enzymol. 152: 432 (1987).* CT (US); Pamela B. Conley, Palo Alto, GenBank Accession No.: CAB01233 (Jun. 20, 2001). CA (US); William M. Grosse, SWALL (SPTR) Accession No.: Q9N4G7 (Oct. 1, 2000). Branford, CT (US); Matthew Hart, San GenBank Accession No.: Z77666 (Jun. 20, 2001). Francisco, CA (US); Ramesh Kekuda, GenBank Accession No. XM 038002 (Oct. 16, 2001). Stamford, CT (US); Richard A. GenBank Accession No. XM 039746 (Oct. 16, 2001). Shimkets, West Haven, CT (US); GenBank Accession No.: AAF51854 (Oct. 4, 2000). Kimberly A. Spytek, New Haven, CT GenBank Accession No.: AAF52569 (Oct. 4, 2000). (US); Edward Szekeres, Jr., Branford, GenBank Accession No.: AAF53188 (Oct. 4, 2000). CT (US); James E. Tomlinson, GenBank Accession No.: AAF55 108 (Oct. 5, 2000). Burlingame, CA (US); James N. GenBank Accession No.: AAF58048 (Oct. 4, 2000). Topper, Los Altos, CA (US); Ruey-Bin GenBank Accession No.: AAF59281 (Oct. 4, 2000). Yang, San Mateo, CA (US) GenBank Accession No.: AE003598 (Oct. 4, 2000). GenBank Accession No.: AE003619 (Oct. 4, 2000). (73) Assignees: Millennium Pharmaceuticals, Inc., GenBank Accession No.: AE003636 (Oct. 4, 2000). Cambridge, MA (US); Curagen GenBank Accession No.: AE003706 (Oct. 5, 2000). Corporation, New Haven, CT (US) GenBank Accession No.: AE003808 (Oct.
    [Show full text]
  • Comparative Sequence Analysis of the South African Vaccine Strain and Two Virulent field Isolates of Lumpy Skin Disease Virus
    Arch Virol (2003) 148: 1335–1356 DOI 10.1007/s00705-003-0102-0 Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of Lumpy skin disease virus P. D. Kara1, C. L. Afonso2, D. B. Wallace1, G. F. Kutish2, C. Abolnik1, Z. Lu2, F. T. Vreede3, L. C. F. Taljaard1, A. Zsak2, G. J. Viljoen1, and D. L. Rock2 1Biotechnology Division, Onderstepoort Veterinary Institute, Onderstepoort, South Africa 2Plum Island Animal Disease Centre, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York, U.S.A. 3IGBMC, Illkirch, France Received November 25, 2002; accepted February 17, 2003 Published online May 5, 2003 c Springer-Verlag 2003 Summary.The genomic sequences of 3 strains of Lumpy skin disease virus (LSDV) (Neethling type) were compared to determine molecular differences, viz. the South African vaccine strain (LW), a virulent field-strain from a recent outbreak in South Africa (LD), and the virulent Kenyan 2490 strain (LK). A comparison between the virulent field isolates indicates that in 29 of the 156 putative genes, only 38 encoded amino acid differences were found, mostly in the variable terminal regions. When the attenuated vaccine strain (LW) was compared with field isolate LD, a total of 438 amino acid substitutions were observed. These were also mainly in the terminal regions, but with notably more frameshifts leading to truncated ORFs as well as deletions and insertions. These modified ORFs encode proteins involved in the regulation of host immune responses, gene expression, DNA repair, host-range specificity and proteins with unassigned functions. We suggest that these differences could lead to restricted immuno-evasive mechanisms and virulence factors present in attenuated LSDV strains.
    [Show full text]
  • Genome-Wide Characterization and Analysis of F-Box Protein-Encoding
    Mol Genet Genomics (2015) 290:1435–1446 DOI 10.1007/s00438-015-1004-z ORIGINAL PAPER Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome Hao-Ran Cui · Zheng-Rong Zhang · Wei lv · Jia-Ning Xu · Xiao-Yun Wang Received: 24 October 2014 / Accepted: 29 January 2015 / Published online: 18 February 2015 © Springer-Verlag Berlin Heidelberg 2015 Abstract The F-box protein family is a large family that Using qRT-PCR to examine the expression of F-box genes is characterized by conserved F-box domains of approxi- encoding proteins with domains related to stress, the results mately 40–50 amino acids in the N-terminus. F-box pro- revealed that F-box proteins were up- or down-regulated, teins participate in diverse cellular processes, such as which suggests that F-box genes were involved in abiotic development of floral organs, signal transduction and stress. The results of this study helped to elucidate the response to stress, primarily as a component of the Skp1- functions of F-box proteins, especially in Rosaceae plants. cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding Keywords Apple · F-box · Bioinformatics · Expression genes (F-box genes for short) were identified and further pattern · Stress subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Introduction Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was The F-box motif is a conserved sequence that is found present with or without the Kelch domain.
    [Show full text]
  • Cul3 Ubiquitin Ligase and Ctb73 Protein Interactions
    Portland State University PDXScholar University Honors Theses University Honors College 2014 Cul3 Ubiquitin Ligase and Ctb73 Protein Interactions Lacey Royer Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/honorstheses Let us know how access to this document benefits ou.y Recommended Citation Royer, Lacey, "Cul3 Ubiquitin Ligase and Ctb73 Protein Interactions" (2014). University Honors Theses. Paper 101. https://doi.org/10.15760/honors.48 This Thesis is brought to you for free and open access. It has been accepted for inclusion in University Honors Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. Cul3 Ubiquitin Ligase and Ctb73 Protein Interactions By Lacey Royer An Undergraduate Thesis Submitted in Partial Fulfillment of the Requirements for the degree of Bachelor of Science in University Honors and Micro/Molecular Biology Thesis Adviser Jeffrey Singer, Ph.D. Portland State University 2014 Table of Contents Page Acknowledgements………………………………………………………………. 3 Introduction…………………………………………………………………………. 4 Methods…………………….………………………………………………………… 10 Results………………………………………………………………………………… 11 Discussion…………………………………………………………………………… 12 Literature Cited………………………………………………………………….. 15 2 Acknowledgements I would like to thank, first and foremost, Dr. Jeff Singer for taking the time to be my thesis advisor and for allowing me to take part in the use of his laboratory and equipment. The experience I have had learning from him and his graduate students was, by far, one of the most rewarding experiences I have had during my college career. A huge and sincere thank you goes to graduate students Brittney Davidge and Jennifer Mitchell; they were so helpful and welcoming. I could not have asked for a better lab group to work with.
    [Show full text]
  • Regulation of Nrf2 by a Keap1-Dependent E3 Ubiquitin Ligase
    REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE A Dissertation presented to The Faculty of the Graduate School at the University of Missouri-Columbia In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by SHIH-CHING (JOYCE) LO Dr. Mark Hannink, Dissertation Adviser DECEMBER 2007 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE Presented by Shih-Ching (Joyce) Lo A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worthy of acceptance. Mark Hannink Thomas Guilfoyle David Pintel Grace Sun Richard Tsika DEDICATION Okay. Mom, I got you the Ph.D. you asked, at the place you picked. Can I now please do something else? Dad, this is to you, too… you accomplice! ACKNOWLEDGEMENTS Dr. Mark Hannink. This work would never have been possible without his guidance, criticism and encouragement. His sheer enthusiasm and critical thinking of science are the most valuable lessons that I could ever obtain during my graduate studies. I should also extend my appreciation to my committee members, Drs. Thomas Guilfoyle, David Pintel, Grace Sun and Richard Tsika, as well as Drs. Lesa Beamer, Joan Conaway, Beverly DaGue, Marc Johnson, Alan Diehl and Michael Henzl, for their insightful advice and thoughtful comments on this work. I thank the present and previous members in Dr. Hannink’s laboratory, including Drs. Donna Zhang, Rick Sachdev, Sang-Hyun Lee, Xuchu Li, as well as Brittany Angle, Carolyn Eberle, Zheng Sun, Jordan Wilkins, Marquis Patrick, Xiaofang Jin, Casey Williams, Joel Pinkston, Julie Unverferth, and Benjamin Creech.
    [Show full text]
  • Keap1 Perceives Stress Via Three Sensors for the Endogenous Signaling Molecules Nitric Oxide, Zinc, and Alkenals
    Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals Michael McMahona,1, Douglas J. Lamontb, Kenneth A. Beattieb, and John D. Hayesa aBiomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland; and bWellcome Trust Building, College of Life Sciences, University of Dundee, Dundee DD1 4HN, Scotland Edited by Mike P. Murphy, Medical Research Council, Cambridge, United Kingdom, and accepted by the Editorial Board September 3, 2010 (received for review June 8, 2010) Recognition and repair of cellular damage is crucial if organisms are tions (12, 13). The problem this diversity of stimuli presents for to survive harmful environmental conditions. In mammals, the Keap1 has not been widely acknowledged because the protein is Keap1 protein orchestrates this response, but how it perceives frequently viewed solely as a sensor of xenobiotic electrophiles. It adverse circumstances is not fully understood. Herein, we implicate has therefore been thought that the ability of such chemicals to NO, Zn2þ, and alkenals, endogenously occurring chemicals whose covalently modify thiol groups provides a satisfactory explanation concentrations increase during stress, in this process. By combining for the action of Keap1 (14). However, although it is true that molecular modeling with phylogenetic, chemical, and functional most thiols in Keap1 become adducted when it is exposed to analyses, we show that Keap1 directly recognizes NO, Zn2þ, and electrophiles in vitro (reviewed in ref. 15), it is unclear whether alkenals through three distinct sensors. The C288 alkenal sensor these modifications occur to a significant extent in vivo. More- is of ancient origin, having evolved in a common ancestor of bila- over, the hypothesis that Keap1 reacts directly with xenobiotic terans.
    [Show full text]
  • Structure of Cul3 in Complex with Vaccinia Virus Protein A55
    bioRxiv preprint doi: https://doi.org/10.1101/461806; this version posted November 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structure of Cul3 in complex with vaccinia virus protein A55 Molecular basis of Cul3 ubiquitin ligase subversion by vaccinia virus protein A55 Chen Gao1, Mitchell A. Pallett1, Tristan I. Croll2, Geoffrey L. Smith1 and Stephen C. Graham1 1Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, United Kingdom; 2Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge CB2 0XY, United Kingdom To whom correspondence should be addressed: Stephen C. Graham: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, United Kingdom; [email protected]; Tel. +44 (0) 1223336920 Keywords: poxvirus, innate immunity, viral immunology, E3 ubiquitin ligase, protein structure, structure-function, isothermal titration calorimetry, X-ray crystallography, BTB-Kelch ABSTRACT BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box based E3 ubiquitin ligases, which mediate protein ubiquitylation leading to proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins, namely A55, C2 and F3. Viruses lacking A55 or C2 demonstrate altered cytopathic effect in cultured cells and altered pathology in vivo. Previous studies show that the ectromelia virus orthologue of A55, EVM150, interacts with Cul3 in cells. We show that A55 binds directly to Cul3 via its N-terminal BTB-BACK domain, and together they form a 2:2 complex in solution. The crystal structure of the A55/Cul3 complex was solved to 2.8 Å resolution.
    [Show full text]
  • Limited Genetic Diversity in the Pvk12 Kelch Protein in Plasmodium Vivax
    Wang et al. Malar J (2016) 15:537 DOI 10.1186/s12936-016-1583-0 Malaria Journal RESEARCH Open Access Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia Meilian Wang1,2*, Faiza Amber Siddiqui2, Qi Fan3, Enjie Luo1, Yaming Cao4 and Liwang Cui1,2* Abstract Background: Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular mark- ers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populations from Southeast Asia, where artemisinin resistance in P. falciparum has emerged. Methods: The PvK12 sequences in 120 P. vivax isolates collected from Thailand (22), Myanmar (32) and China (66) between 2004 and 2008 were obtained and 353 PvK12 sequences from worldwide populations were retrieved for further analysis. Results: These PvK12 sequences revealed a very low level of genetic diversity (π 0.00003) with only three single nucleotide polymorphisms (SNPs). Of these three SNPs, only G581R is nonsynonymous.= The synonymous mutation S88S is present in 3% (1/32) of the Myanmar samples, while G704G and G581R are present in 1.5% (1/66) and 3% (2/66) of the samples from China, respectively. None of the mutations observed in the P. vivax samples were associ- ated with artemisinin resistance in P. falciparum. Furthermore, analysis of 473 PvK12 sequences from twelve worldwide P.
    [Show full text]
  • Comparative Structural and Evolutionary Analyses Predict
    www.nature.com/scientificreports OPEN Comparative structural and evolutionary analyses predict functional sites in the artemisinin Received: 11 March 2019 Accepted: 3 July 2019 resistance malaria protein K13 Published: xx xx xxxx Romain Coppée1, Daniel C. Jefares2, Maria A. Miteva3, Audrey Sabbagh1 & Jérôme Clain1,4 Numerous mutations in the Plasmodium falciparum Kelch13 (K13) protein confer resistance to artemisinin derivatives, the current front-line antimalarial drugs. K13 is an essential protein that contains BTB and Kelch-repeat propeller (KREP) domains usually found in E3 ubiquitin ligase complexes that target substrate protein(s) for ubiquitin-dependent degradation. K13 is thought to bind substrate proteins, but its functional/interaction sites and the structural alterations associated with artemisinin resistance mutations remain unknown. Here, we screened for the most evolutionarily conserved sites in the protein structure of K13 as indicators of structural and/or functional constraints. We inferred structure-dependent substitution rates at each amino acid site of the highly conserved K13 protein during the evolution of Apicomplexa parasites. We found two solvent-exposed patches of extraordinarily conserved sites likely involved in protein-protein interactions, one in BTB and the other one in KREP. The conserved patch in K13 KREP overlaps with a shallow pocket that displays a diferential electrostatic surface potential, relative to neighboring sites, and that is rich in serine and arginine residues. Comparative structural and evolutionary analyses revealed that these properties were also found in the functionally-validated shallow pocket of other KREPs including that of the cancer-related KEAP1 protein. Finally, molecular dynamics simulations carried out on PfK13 R539T and C580Y artemisinin resistance mutant structures revealed some local structural destabilization of KREP but not in its shallow pocket.
    [Show full text]