Določanje aktivnosti in proteinskih nivojev katalitičnih podenot proteasomov v trombocitih zdravih darovalcev

Grosek, Vanja (avtor), Gobec, Martina (mentor), Smrdel, Lara (komentor)

Proteasom je večkatalitični proteazni kompleks, ki omogoča razgradnjo proteinov. Obstaja v več strukturnih oblikah in tkivno specifičnih različicah. Najbolj pogosta oblika je konstitutivni proteasom ter imunoproteasom. Nedavne raziskave so pokazale, da se proteasomi nahajajo tudi v trombocitih, pri čemer je njihova vloga precej neznana. Trombociti se lahko glede na posamezne lastnosti med posamezniki močno razlikujejo. Na podlagi tega smo sklepali, da obstajajo razlike med posamezniki tudi v izražanju in aktivnosti posameznih katalitičnih podenot proteasoma. Našo domnevo smo preverjali na vzorcih naključnih darovalcev, katerim smo določili proteinske nivoje standardnih in imunoproteasomskih katalitičnih podenot v trombocitih s prenosom western. Rezultati so pokazali razlike med posamezniki, pri tem pa smo opazili, da so razlike večje med izražanjem imunoproteasomskih kot konstitutivnih podenot. Istim vzorcem darovalcev smo nato s sondami za aktivnost določili še katalitično aktivnost posamezne podenote, kjer smo prav tako opazili razlike in višjo aktivnost imunoproteasomskih podenot β1i in β5i kot konstitutivnih podenot β1c in β5c, kar potrjujejo tudi podatki iz literature. V nadaljevanju smo preučevali vpliv selektivnih zaviralcev katalitičnih podenot imunoproteasoma, kjer smo za zaviranje ß1i podenote uporabili KZR-504, za zaviranje ß5i podenote pa LMP7-IN-1. Pri tem se je izkazalo, da 50 nM koncentracija obeh selektivnih zaviralcev po 3-urni izpostavitvi povzroči skoraj popolno zaviranje β1i oziroma β5i podenote. To koncentracijo zaviralcev smo nato uporabili pri naslednjem poskusu, kjer nas je zanimalo, ali aktivacija trombocitov z adenozin difosfatom (ADP) kakorkoli vpliva na aktivnost podenot imunoproteasoma. Ugotovili smo, da ADP pod danimi pogoji ne vpliva na katalitično aktivnost β1i ali β5i podenote proteasoma. Nadalje smo preverili, ali zaviranje imunoproteasoma vpliva na aktivacijo trombocitov. S tem namenom smo s pretočno citometrijo določili sproščanje izbranih kemokinov iz trombocitnih granul. Pri tem smo ugotovili, da vodi aktivacija trombocitov z ADP v povečano sproščanje kemokina RANTES, vendar prisotnost zaviralcev KZR-504 in LMP7-IN-1 ne vpliva statistično značilno na njegov nivo. Rezultati kažejo na razlike v proteinskem nivoju in aktivnosti katalitičnih podenot proteasoma v trombocitih posameznikov. Katalitične podenote lahko uspešno zaviramo že po nekajurni izpostavitvi selektivnim zaviralcem. Potrebne so nadaljnje študije za osvetlitev vloge proteasomov v procesu aktivacije trombocitov.

• Jezik: Slovenski jezik
• Ključne besede: imunoproteasom, trombociti, aktivnost, selektivni zaviralci, RANTES
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-156222
• Datum objave v RUL: 15.05.2024

GROSEK, Vanja, 2024, Določanje aktivnosti in proteinskih nivojev katalitičnih podenot proteasomov v trombocitih zdravih darovalcev [na spletu]. Magistrsko delo. [Dostopano 5 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=156222

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determining the activity and protein levels of catalyticaly active proteasome subunits in platelets of healthy donors

Izvleček

Proteasome is a multi-catalytic protease complex that enables protein degradation. It exists in several structural forms and tissue-specific variants. The most common is the constitutive proteasome and immunoproteasome, which were recently also found to be present in platelets. It is known that platelets and their characteristic can heavily vary among individuals. Based on this, we hypothesized that there are also differences between individuals in the expression and activity of individual catalytic subunits of the proteasome. Our hypothesis was tested in samples from random donors, in whom we determined the protein levels of constitutive and immunoproteasome catalytic subunits in platelets by western blotting. The results showed inter-individual differences, with a greater variation in the expression of immunoproteasome than constitutive subunits. The same donor samples were then subjected to activity based probe assays to determine the activity of the individual subunits in platelets. Differences were also observed in this parameter, where higher activity of the immunoproteasomal subunits β1i and β5i than of the β1c and β5c subunits. We further investigated the effect of selective inhibitors of the catalytic subunits of the immunoproteasome, using KZR-504 to inhibit the β1i subunit and LMP7-IN-1 to inhibit the β5i subunit. Here, 50 nM concentrations of the two selective inhibitors were shown to result in almost complete inhibition of the corresponding subunits after 3 hours. This concentration of inhibitors was then used in a further experiment, where we were interested to see whether platelet activation by adenosine diphosphate (ADP) had any effect on the activity of the immunoproteasome subunits in the presence of selective inhibitors. We found that under the given conditions, ADP did not affect the catalytic activity of the β1i or β5i subunits. We then tested whether inhibition of the immunoproteasome affects platelet activation by determining the concentration of released chemokines from the platelet granules. We found that platelet activation with ADP leads to an increased release of the chemokine RANTES, however the presence of the inhibitors KZR-504 and LMP7-IN-1 did not cause any statistically relevant changes. The results show differences in the protein levels and activities of the proteasome catalytic subunits in platelets between individuals. The catalytic subunits can be successfully inhibited after only a few hours of exposure to selective inhibitors. Further studies are needed to investigate the role of the proteasome in the process of platelet activation.

• Ključne besede: immunoproteasome, platelets, activity, selective inhibitors, RANTES