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Proučevanje medsebojnega uravnavanja proteasomov in katepsinov v aktivirani mikrogliji
ID Bratušek, Tjaša (Avtor), ID Pišlar, Anja (Mentor) Več o mentorju... Povezava se odpre v novem oknu, ID Gobec, Martina (Komentor)

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Izvleček
Natančen nadzor dveh glavnih sistemov proteolize, ubikvintin-proteasomskega sistema in avtofagijsko-lizosomske poti, je pomemben za normalno delovanje celic. S staranjem proteasom oslabi, kar povzroča kopičenje proteinov in tveganje za nevrodegeneracijo, medtem ko njegova alternativna oblika, imunoproteasom, postane aktivnejša. Prekomerna aktivnost imunoproteasoma lahko sčasoma škoduje nevronom in spodbuja vnetja, kar lahko prispeva k razvoju nevrodegenerativnih stanj. Znano pa je, da imajo pomembno vlogo na potek teh stanj tudi katepsini, ki so del lizosomske poti. Zato smo v sklopu magistrske naloge preverili vpliv medsebojnega uravnavanja proteasoma in katepsinov v aktiviranih celicah mikroglije. Pričeli smo s postavitvijo celičnega modela polarizirane mikroglije z uporabo celične linije BV2, pri katerem smo pokazali, da stimulacija z lipopolisaharidom (LPS) in interferonom gama (INF-γ) vodi v polarizacijo mikroglije v smeri M1 fenotipa. Polarizacijo mikroglije v pro-vnetni fenotip smo nadalje potrdili z merjenjem količine sproščenega dušikovega oksida. V nadaljevanju smo preučevali vpliv stimulacije celic BV2 na znotrajcelično aktivnost katepsina X in S, pri čemer smo ugotovili, da tako LPS kot INF-γ povzročita znižanje encimske aktivnosti. Hkrati smo preverili tudi vpliv stimulacije na izražanje β5 in β1 podenote proteasoma in imunoproteasoma. Sočasna stimulacija celic BV2 z LPS in INF-γ ni znatno vplivala na izražanje β5 in β1 podenote konstitutivnega proteasoma, medtem ko je prišlo do izrazitega povečanja njegove alternativne oblike. V zadnjem sklopu magistrske naloge pa smo nadalje ovrednotili vpliv sočasnega zaviranja katepsina X oz. S in β podenote (imuno)proteasoma na polarizacijo celic BV2 v M1 fenotip. Najbolj obetavne rezultate smo dosegli s sočasno uporabo ireverzibilnega zaviralca katepsina X, AMS36, in selektivnega zaviralca β5i podenote imunoproteasoma, KZR-504, saj sta slednja preprečila polarizacijo mikroglije v smeri M1 fenotipa, kar je bilo razvidno iz zmanjšanega deleža nastalega NO. Znižane ravni NO smo dosegli tudi s tretiranjem celic z neselektivnim zaviralcem proteasoma, bortezomibom, vendar je slednji izkazal citotoksičnost za celice mikroglije. Iz dobljenih rezultatov lahko zaključimo, da zaviranje aktivnosti katepsinov kot tudi imunoproteasoma predstavlja obetavno strategijo za zdravljenje in preprečevanje nevrodegenerativnih bolezni, povezanih s prekomernim vnetjem.

Jezik:Slovenski jezik
Ključne besede:mikroglija, katepsin X, katepsin S, (imuno)proteasom, vnetje
Vrsta gradiva:Magistrsko delo/naloga
Organizacija:FFA - Fakulteta za farmacijo
Leto izida:2024
PID:20.500.12556/RUL-155696 Povezava se odpre v novem oknu
Datum objave v RUL:12.04.2024
Število ogledov:337
Število prenosov:89
Metapodatki:XML DC-XML DC-RDF
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Sekundarni jezik

Jezik:Angleški jezik
Naslov:Study of the interplay of proteasomes and cathepsins in activated microglia
Izvleček:
The precise control of two major proteolysis systems, the ubiquintin-proteasome system and the autophagy-lysosomal pathway, is important for normal cell function. With aging, the proteasome weakens, causing protein accumulation and the risk of neurodegeneration, while its alternative form, the immunoproteasome, becomes more active. Excessive immunoproteasome activity can eventually damage neurons and promote inflammation, which may contribute to the development of neurodegenerative conditions. However, it is known that cathepsins, which are part of the lysosomal pathway, also play an important role in the course of these conditions. Therefore, as part of the master's thesis, we checked the influence of mutual regulation of the proteasome and cathepsins in activated microglia cells. We started by establishing a cell model of polarized microglia using the BV2 cell line, in which we showed that stimulation with lipopolysaccharide (LPS) and interferon gamma (INF-γ) leads to the polarization of microglia towards the M1 phenotype. The polarization of microglia into a pro-inflammatory phenotype was further confirmed by measuring the amount of released nitric oxide. Next, we studied the influence of stimulation of BV2 cells on the intracellular activity of cathepsin X and S, and we found that both LPS and INF-γ cause a decrease in enzyme activity. At the same time, we also checked the influence of stimulation on the expression of the β5 and β1 subunits of the proteasome and the immunoproteasome. Simultaneous stimulation of BV2 cells with LPS and INF-γ did not significantly affect the expression of the β5 and β1 subunits of the constitutive proteasome, while there was a marked increase of its alternative form. In the last part of the master's thesis, we further evaluated the influence of the simultaneous inhibition of cathepsin X or S and β subunits of the (immuno)proteasome on the polarization of BV2 cells to the M1 phenotype. The most promising results were achieved with the simultaneous use of an irreversible cathepsin X inhibitor, AMS36, and a selective inhibitor of the β5i subunit of the immunoproteasome, KZR-504, as the latter prevented the polarization of microglia towards the M1 phenotype, which was evident from the reduced proportion of NO produced. Reduced levels of NO were also achieved by treating cells with the non-selective proteasome inhibitor, bortezomib, but the latter showed cytotoxicity for mircoglia cells. From the obtained results, we can conclude that inhibiting the activity of cathepsins, such as the immunoproteasome, represents a promising strategy for the treatment and prevention of neurodegenerative diseases associated with excessive inflammation.

Ključne besede:microglia, cathepsin X, cathepsin S, (immuno)proteasome, inflammation

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