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Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867614077852844032 |
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| access_status_str | Open Access |
| author | Caillet, Céline |
| author2 | Zininga, Tawanda |
| author_browse | Caillet, Céline Zininga, Tawanda |
| author_facet | Zininga, Tawanda Caillet, Céline |
| author_sort | Caillet, Céline |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023.
|
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129370 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:46:18.613Z |
| 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:10019.1/129370 Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones Caillet, Céline Zininga, Tawanda De Villiers, Marianne Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Malaria -- Pathogenesis Plasmodium falciparum Heat shock proteins Molecular chaperones -- Mechanism of action Protein folding Protein-protein interactions -- Regulation -- Molecular aspects UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: The Plasmodium falciparum parasite is the most lethal form of malaria known to be infectious to humans and is responsible for the majority of malaria related deaths. The survival of the parasites throughout their complex life cycle inside both the mosquito vector and the human host, is dependent on the action of various molecular chaperones. Heat shock proteins (Hsps) are a group of molecular chaperones that regulate several cellular processes within the parasite. Most importantly, they play an essential role in maintaining the proteome integrity within P. falciparum, by facilitating the folding of proteins into their functional structures. This is required to maintain host infectivity, mediate malaria pathogenicity and enable parasitic survival. The major cytosolic chaperones, P. falciparum Hsp90 (PfHsp90) and Hsp70-z (PfHsp70-z), are known for their crucial function in the growth, development and survival of the parasite, particularly in the intra-erythrocytic stage of their life cycle within the human host. Hsp90 also collaborates with a variety of co-chaperone and chaperone partner proteins that assist in their functional protein folding cycle. Although the functional partnerships with Hsp90 have been extensively studied in eukaryotic organisms, they are not well established in the protozoan parasites. Therefore, targeting the parasite’s robust protein quality control mechanism imperative for their survival, is challenging. The main aim of this study was to establish and characterise the chaperone complex association between the cytosolic PfHsp90 and PfHsp70-z proteins. Recombinant full-length PfHsp90 and PfHsp70-z were expressed in Escherichia coli XL1 Blue cells and purified using immobilised metal affinity chromatography and size exclusion chromatography. Bioinformatics analysis predicted a direct association between PfHsp90 and PfHsp70-z, which was also validated in vitro, for the first time, through surface plasmon resonance. In addition, their direct interaction displayed slightly greater binding kinetics in a nucleotide-free state when compared to their nucleotide-bound state in the presence of ATP or ADP. Thermal stability analysis of PfHsp90 and PfHsp70-z revealed that both proteins were resilient to heat stress above 50°C, which corresponds to their heat shock protein characteristic of withstanding extreme physiological stressors. Interestingly, it was also observed that the thermal stability of the chaperone proteins were further enhanced when complexed together. The functionality of the complex association was further elucidated by assessing their ability to suppress the aggregation of a client protein namely, malate dehydrogenase (MDH). The chaperone complex was found to efficiently suppress the aggregation of the client protein by ~80%. However, it was also noted that the independent function of PfHsp90 was more effective at suppressing the aggregation of MDH when compared to the complex. Overall, this study provided the first evidence of a direct and functional association between cytosolic PfHsp90 and PfHsp70-z. These findings suggest that the chaperone complex performs a cytoprotective role during the development and survival of the P. falciparum parasite throughout their life cycle. Therefore, further elucidating the parasite’s protein folding machinery and their functional partnerships, may provide novel targets that can be utilised to weaken their protective mechanism and help combat malaria. AFRIKAANSE OPSOMMING: Die Plasmodium falciparum parasiet is die dodelikste vorm van malaria wat bekend is en is verantwoordelik vir die meeste malariaverwante sterftes in die wêreld. Die oorlewing van die parasiete gedurende hul komplekse lewensiklus binne beide die muskietvektor en die menslike gasheer, is afhanklik van die werking van verskillende molekulêre chaperones. Hitteskokproteïene (Hsps) is 'n groep molekulêre chaperones wat verskeie sellulêre prosesse binne die parasiet reguleer. Die belangrikste is dat hulle 'n noodsaaklike rol speel in die handhawing van die proteoomintegriteit binne P. falciparum, deur die vou van proteïene in hul funksionele strukture te vergemaklik. Dit is nodig om gasheerinfektiwiteit te handhaaf, malariapatogenisiteit te bemiddel en parasitiese oorlewing moontlik te maak. Die belangrikste sitosoliese chaperones, P. falciparum Hsp90 (PfHsp90) en Hsp70-z (PfHsp70-z), is bekend vir hul belangrike funksie in die groei, ontwikkeling en oorlewing van die parasiet, veral in die intra-eritrositiese stadium van hul lewensiklus binne die menslike gasheer. Hsp90 werk ook saam met 'n verskeidenheid mede-chaperone- en chaperone-vennootproteïene wat help met hul funksionele proteïenvousiklus. Alhoewel die funksionele vennootskappe met Hsp90 omvattend in eukariotiese organismes bestudeer is, is hulle nie goed gevestig in die protosoë parasiete nie. Daarom is dit 'n uitdaging om die parasiet se robuuste proteïengehaltebeheermeganisme te teiken wat noodsaaklik is vir hul oorlewing. Die hoofdoel van hierdie studie was om die chaperone komplekse assosiasie tussen die sitosoliese PfHsp90 en PfHsp70-z proteïene vas te stel en te karakteriseer. Rekombinante vollengte PfHsp90 en PfHsp70-z is uitgedruk in E. coli XL1 Blou selle en gesuiwer met behulp van geïmmobiliseerde metaal affiniteit chromatografie en grootte uitsluiting chromatografie. Bioinformatika-analise het 'n direkte verband tussen PfHsp90 en PfHsp70-z voorspel, wat ook vir die eerste keer in vitro gevalideer is deur oppervlakplasmonresonansie. Daarbenewens het hul direkte interaksie groter bindende kinetika in 'n nukleotiedvrye toestand getoon in vergelyking met hul nukleotiedgebonde toestand in die teenwoordigheid van ATP of ADP. Termiese stabiliteitsanalise van PfHsp90 en PfHsp70-z het aan die lig gebring dat albei proteïene bestand was teen hittestres bo 50°C, wat ooreenstem met hul hitteskokproteïen wat kenmerkend is van die weerstand teen uiterste fisiologiese stressors. Interessant genoeg was dit ook waargeneem dat die termiese stabiliteit van die chaperone proteïene verder verbeter is wanneer hulle saam gekompleks is. Die funksionaliteit van die komplekse assosiasie is verder toegelig deur hul vermoë om die samevoeging van 'n kliëntproteïen te onderdruk, naamlik malaatdehidrogenase (MDH), te beoordeel. Daar is gevind dat die chaperone- kompleks die samevoeging van die kliëntproteïen doeltreffend onderdruk met ~80%. Daar is egter ook opgemerk dat die onafhanklike funksie van PfHsp90 meer effektief was om die samevoeging van MDH te onderdruk in vergelyking met die kompleks. Oor die algemeen het hierdie studie die eerste bewys gelewer van 'n direkte en funksionele verband tussen sitosoliese PfHsp90 en PfHsp70-z. Hierdie bevindings daarop dat die chaperone-kompleks 'n sitoprotektiewe rol speel tydens die ontwikkeling en oorlewing van die P. falciparum parasiet gedurende hul lewensiklus. Daarom kan die verdere toeligting van die parasiet se proteïenvoumasjinerie en hul funksionele vennootskappe nuwe teikens bied wat gebruik kan word om hul beskermingsmeganisme te verswak en malaria te help bekamp. Masters 2023-11-17T14:04:06Z 2024-02-20T09:11:39Z 2023-11-17T14:04:06Z 2024-02-20T09:11:39Z 2023-12 Thesis https://scholar.sun.ac.za/handle/10019.1/129370 en Stellenbosch University xvi, 95 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Malaria -- Pathogenesis Plasmodium falciparum Heat shock proteins Molecular chaperones -- Mechanism of action Protein folding Protein-protein interactions -- Regulation -- Molecular aspects UCTD Caillet, Céline Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title | Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title_full | Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title_fullStr | Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title_full_unstemmed | Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title_short | Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones |
| title_sort | characterisation of the cytosolic plasmodium falciparum hsp90 and hsp70 z chaperones |
| topic | Malaria -- Pathogenesis Plasmodium falciparum Heat shock proteins Molecular chaperones -- Mechanism of action Protein folding Protein-protein interactions -- Regulation -- Molecular aspects UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/129370 |
| work_keys_str_mv | AT cailletceline characterisationofthecytosolicplasmodiumfalciparumhsp90andhsp70zchaperones |