Deubiquitination : does the answer to cardiotoxicity lie within the ubiquitin proteasome pathway

Ogundipe, Temitope R. (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Introduction: Cardiotoxicity, a complication that arises from anthracycline use is one that has confounded scientists for decades. Attempts have been made to attenuate the development of this condition through the use of anti-oxidants with little success and this has led to calls for new adjuvant therapies. One area that has been identified as a potential intervention involves the ubiquitin proteasome system (UPS) and its regulation and degradation of proteins that control mitochondrial morphology, apoptosis and cellular anti-oxidants. This process can be reversed through the use of de-ubiquitinating enzymes (DUBs); however their role in this context is relatively unknown. Therefore, this study aimed to investigate the role of specific DUBs relevant in this context and whether the manipulation of their protein expression levels will be beneficial. Methods: Chronic doxorubicin (DOX)-induced toxicity was induced in H9C2 cardiomyoblasts and male Sprague-Dawley rats for 120 hrs (0.2 μM) and eight weeks (2.5 mg/kg/week) respectively. Baseline protein expression of DUBs as well as their down-stream factors was determined by western blotting on both models. Immunocytochemistry was undertaken only for in vitro studies. DUBs were down-regulated using SiRNA, and the subsequent effect on downstream proteins was determined through western blotting. Mitochondrial morphology was evaluated by fluorescence microscopy, while cellular toxicity and ATP production were assessed using a mitochondrial toxicity assay. Results and Discussion: DOX increased the expression of USP9x (103.7 ± 4.7%, p<0.01), which regulates MCL1 (long-fragment), an anti-apoptotic protein which was down-regulated in this scenario. Interestingly, the pro-apoptotic short-fragment of MCL1 was up-regulated, suggesting a mechanism by which DOX uses USP9x to promote apoptosis. DOX treatment also reduced USP30 expression (27.5 ± 3.7%, p<0.01), as well as its downstream target, the mitofusin proteins (22.7 ± 5.9%, p<0.001) which regulate mitochondrial fusion during mitochondrial dynamics. USP36 showed little variation between the two groups however, DOX reduced SOD2 expression (250.9 ± 6.8%, p<0.001). While both models utilised produced similar results, there was minor variation in the results. When DUB (SiRNA) was initiated in the presence of DOX, mitochondrial morphology appeared to improve. Interestingly, while the known-down of some DUBs (USP30 and USP36) did not modify mitochondrial toxicity except when USP9x was abolished, ATP synthesis was significantly upregulated in all intervention groups when compared to DOX treatment alone. Although more research into this topic is urgently needed, it is clear from the positive results obtained above that de-ubiquitination may be a mechanism that can be exploited as a potential treatment strategy in this context.

AFRIKAANSE OPSOMMING: Inleiding: Kardiotoksisiteit, as gevolg van antrasiklien gebruik, is ‘n komplikasie wat wetenskaplikes vir dekades verwar. Antioksidante is gebruik om die ontwikkelling van die toestand te probeer onderdruk, maar pogings was onsuksesvol en het aanleiding daartoe gegee dat nuwe ondersteuningsterapiëe ontwikkel moes word. Een area wat geteiken is vir potensiële intervensies sluit die ubikwitienproteosoomsisteem (UPS) in, en die regulering en degradering van proteïene wat mitochondriale morfologie, aptoptose en sellulêre antioksidante beheer. Hierdie proses is omkeerbaar deur die gebruik van die de-ubikwitieneringsensiem (DUBs), maar die rol in hierdie konteks is onduidelik. Gevolglik het hierdie studie daarin gepoog om die rol van spesifieke DUBs wat in hierdie konteks verwant is, asook of die manipulering van die proteïenuitdrukkingsvlakke voordelig sal wees te ondersoek. Metodes: H9C2 kardiomioblaste en manlike Sprague-Dawley rotte is onderskeidelik aan 120 uur (0.2 μM) en agt weke (2.5 mg/kg/week) van chroniese doksorubisien (DOX)-geïnduseerde toksisiteit blootgestel. Basislyn DUBs proteïenuitdrukking, asook hulle afstroomfaktore is deur middel van Westerse blattering op beide modelle, en slegs met behulp van immunositochemie in vitro bepaal. DUBs is afgereguleer deur van SiRNA gebruik te maak, waarna die gevolglike effekte van die afstroomproteïene deur middel van Westerse blattering bepaal is. Mitochondriale morfologie is deur middel van fluoresensie mikroskopie ondersoek, terwyl sellulêre toksisiteit en ATP produksie deur middel van ‘n mitochondriale toksisiteittoets bepaal is. Resultate en Bespreking: DOX het die uitdrukking van USP9x (103.7 ± 4.7%, p<0.01) verhoog wat MCL1 (langfragment) reguleer. Laasgenoemde is ‘n anti-apoptotiese proteïen wat afgereguleer is in hierdie scenario. Die pro-apoptotiese kortfragment van MCL1 was merkwaardig opgereguleer, wat moontlik ‘n meganisme kan beskryf waarin DOX, USP9x gebruik om apoptose te stimuleer. DOX behandeling het ook USP30 uitdrukking verlaag (27.5 ± 3.7%, p<0.01), asook die afstroomteiken, mitofusienproteïene (22.7 ± 5.9%, p<0.001) wat mitochondriale fusie tydens mitochondriale dinamika reguleer. Hoewel USP36 min variasie tussen die twee groepe getoon het, het DOX SOD2 uitdrukking verlaag (250.9 ± 6.8%, p<0.001). Beide modelle het soorgelyke resultate opgelewer, maar daar was min variasie in die resultate. Nadat DUB (SiRNA) in die teenwoordigheid van DOX geïnisieer is, blyk dit dat die mitochondriale morfologie verbeter het. Terwyl die onderdrukking van sommige DUBs (USP30 en USP36) nie die mitochondriale toksisiteit kon modifiseer nie, behalwe as USP9x afgeskakel was, was ATP sintese interresant genoeg betekenisvol opgereguleer in alle intervensie groepe in vergelyking met die DOX alleen groep. Alhoewel daar nog baie navorsing gedoen moet word op hierdie onderwerp, is dit duidelik uit hierdie positiewe resultate dat die de-ubikwitienasie moontlik as megansime ondersoek kan word vir die potensiële behandelingsstrategie in hierdie konteks.

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