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Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins
Thesis (PhD)--Stellenbosch University, 2023.
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| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613818815774720 |
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| access_status_str | Open Access |
| author | De Buys, Keren |
| author2 | Kinnear, Craig John |
| author_browse | De Buys, Keren Kinnear, Craig John |
| author_facet | Kinnear, Craig John De Buys, Keren |
| author_sort | De Buys, Keren |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129317 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:42:11.774Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/129317 Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins De Buys, Keren Kinnear, Craig John Ramharack, Pritika Loos, Ben Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Intracellular pathogens Mycobacterium tuberculosis Proteolysis Host-parasite relationships UCTD Thesis (PhD)--Stellenbosch University, 2023. ENGLISH ABSTRACT: The intracellular pathogen Mycobacterium tuberculosis, transmitted via aerosol droplets, is the causative agent of tuberculosis. The autophagic degradation of intracellular pathogens, such as M. tuberculosis is known as xenophagy, where intracellular proteins are targeted for degradation by ubiquitin. Ubiquitin is a 76 amino acid protein that is ubiquitously expressed in all eukaryotic cells and is recruited to tag unwanted cellular material for degradation. The post-translational modification of a protein by ubiquitin is catalysed by an enzyme cascade consisting of an E1 ubiquitin activating enzyme, E2 conjugating enzyme and E3 ubiquitin ligase. E3 ubiquitin ligases are responsible for recognising specific M. tuberculosis proteins and tagging them for degradation. In addition to Parkin, Smurf1 and Nedd4 have been found to co-localize with M. tuberculosis and modulate the intercellular survival of M. tuberculosis. Smurf1 recognizes proteins that contain a P-P-x-Y recognition domain via its WW2 domain; while Nedd4 recognizes proteins that contain either a P-P-x-Y or L-P-x-Y recognition domain via its WW3 domain. However, the specific proteins that are recognised by Smurf1 and Nedd4 have not been investigated. This study thus aimed to investigate and identify the specific mycobacterial proteins that are ubiquitinated by the host E3 ubiquitin ligases Smurf1 and Nedd4, using molecular modelling, molecular docking, and molecular dynamic simulations. Mycobacterial proteins that contain the recognition motifs of Smurf1 and Nedd4 were searched on ProSite, and filtered for expression by M. tuberculosis, subcellular localization, and the presence of lysine residues in the amino acid sequence. Crystal structures were obtained from the Protein Data Bank or homologous sequences were used for homology modelling using the Modeller software add-on in UCSF CHIMERA-Tools. The crystal structures or modelled structures were used for molecular docking using the online server HDock. Molecular dynamic simulations were then performed to determine whether Smurf1 and Nedd4 recognize and interact with the identified mycobacterial proteins. Based on molecular dynamic simulation analysis, Smurf1 was found to optimally interact with rv0126 and Rv0189c; Nedd4 was found to interact with the P-P-x-Y motif interacting mycobacterial proteins Rv0126 and Rv0436c, and with the L-P-x-Y motif interacting with mycobacterial proteins Rv0824c, Rv1161, Rv1415, and Rv1477. Autophagy’s role in eliminating M. tuberculosis and the proteins involved represent promising candidates for host-based therapeutics. Thus, knowledge regarding the host E3 ubiquitin ligases involved and the mycobacterial proteins that are ubiquitinated will contribute to the understanding of the ubiquitin-mediated clearance of M. tuberculosis and potentially aid the design of effective host-based therapeutics. This study therefore represents the first steps in identifying both the host E3 ubiquitin ligases involved and the mycobacterial proteins that are potentially ubiquitinated for the autophagic clearance of M. tuberculosis. AFRIKAANSE OPSOMMING: Die intrasellulêre patogeen M. tuberculosis, wat deur aërosoldruppels oorgedra word, is die veroorsakende middel van tuberkulose. Die outofagiese afbraak van intrasellulêre patogene, soos M. tuberculosis, staan bekend as xenophagy. Ubiquitine is 'n 76 aminosuurresidu wat alomteenwoordig uitgedruk word in alle eukariotiese selle en word gewerf om ongewenste sellulêre materiaal vir afbraak te merk. Die post-translasionele modifikasie van 'n proteïen deur ubiquitine word gekataliseer deur 'n ensiemkaskade wat bestaan uit 'n E1-ubiquitine aktiverende ensiem, E2-vervoegende ensiem en E3-ubiquitine ligase. E3 ubiquitine ligase is verantwoordelik vir die herkenning van spesifieke M. tuberculosis proteïene en merk hulle vir afbraak. Benewens Parkin, is dit gevind dat Smurf1 en Nedd4 saam met M. tuberculosis lokaliseer en die intersellulêre oorlewing van M. tuberculosis moduleer. Smurf1 herken proteïene wat 'n P-P-x-Y-herkenningsdomein bevat deur sy WW2-domein; terwyl Nedd4 proteïene herken wat óf 'n P-P-x-Y- óf L-P-x-Y-herkenningsdomein bevat deursy WW3-domein. Die spesifieke proteïene wat deur Smurf1 en Nedd4 erken word, is egter nie ondersoek nie. Hierdie studie het dus die doel gehad om die spesifieke mikobakteriese proteïene wat deur die gasheer E3 ubiquitine ligase Smurf1 en Nedd4 alomteenwoordig is, te ondersoek en te identifiseer, met behulp van molekulêre modellering, molekulêre dok en molekulêre dinamiese simulasies. Mikobakteriese proteïene wat die herkenningsmotiewe van Smurf1 en Nedd4 bevat, is op ProSite gesoek en gefiltreer vir uitdrukking deur M. tuberculosis, subsellulêre lokalisering en die voorwendsel van lysienresidu in die aminosuurvolgorde. Kristalstrukture is verkry uit die Protein Data Bank of homoloë rye is gebruik vir homologiemodellering met behulp van die Modeller-sagteware-byvoeging in UCSF CHIMERA-Tools. Voordat molekulêre dok met behulp van die aanlynbediener HDock, is die strukture voorberei met behulp van Dockprep. Molekulêre dinamiese simulasies is daarna uitgevoer met behulp van die grafiese verwerkingseenheidweergawe van deeltjiemaas Ewald molekulêre dinamika om te bepaal of Smurf1 en Nedd4 die geïdentifiseerde mikobakteriese proteïene herken en daarmee in wisselwerking tree. Die trajekte is dan ontleed deur wortelgemiddelde vierkantafwyking, wortelgemiddelde kwadraatskommeling, radius van gyrasie, vrye bindingsenergie en waterstofbindingsvorming. Op grond van wortelgemiddelde vierkante afwyking, wortelgemiddelde kwadraatskommeling, waterstofbindings stabiliteit, radius van gyrasie, oplosmiddel toeganklike oppervlakte, vrye bindingsenergie en die vorming van waterstofbindings met lisienresidu in die mikobakteriese proteïene, is dit gevind dat Smurf1 met rv0126 en rv0189c interak; Nedd4 met die P-P-x-Y-motief wat mikobakteriese proteïene Rv0126 en Rv0436c bevat interak, en met die L-P-x-Y-motief wat mikobakteriese proteïene Rv0824c, Rv1161, Rv1415 en Rv1477 bevat interak. Outofagie se rol in die uitskakeling van M. tuberculosis en die betrokke proteïene verteenwoordig belowende kandidate vir gasheergebaseerde terapeutiese middels. Dus, kennis rakende die gasheer E3 ubiquitine ligase betrokke en die mikobakteriese proteïene wat alomteenwoordig is, sal bydra tot die begrip van die ubiquitine-gemedieerde klaring van M. tuberculosis en moontlik die ontwerp van effektiewe gasheergebaseerde terapeutiese middels help. Hierdie studie verteenwoordig dus die eerste stappe in die identifisering van beide die gasheer E3 ubiquitin ligases betrokke en die mikobakteriese proteïene wat moontlik ubiquitine is vir die outofagiese klaring van M. tuberculosis. Doctoral 2023-08-29T09:18:46Z 2024-02-20T07:20:14Z 2023-08-29T09:18:46Z 2024-02-20T07:20:14Z 2023-08 Thesis https://scholar.sun.ac.za/handle/10019.1/129317 en Stellenbosch University xxiii, 137 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Intracellular pathogens Mycobacterium tuberculosis Proteolysis Host-parasite relationships UCTD De Buys, Keren Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title | Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title_full | Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title_fullStr | Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title_full_unstemmed | Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title_short | Elucidating the Smurf1- and other E3 ligase-mediated ubiquitination of mycobacterial proteins |
| title_sort | elucidating the smurf1 and other e3 ligase mediated ubiquitination of mycobacterial proteins |
| topic | Intracellular pathogens Mycobacterium tuberculosis Proteolysis Host-parasite relationships UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/129317 |
| work_keys_str_mv | AT debuyskeren elucidatingthesmurf1andothere3ligasemediatedubiquitinationofmycobacterialproteins |