[magistrsko delo]
Toni Nagode (Avtor), Toni Petan (Mentor), Brigita Lenarčič (Član komisije za zagovor), Igor Križaj (Član komisije za zagovor), Miha Pavšič (Komentor)

Povzetek

Sekretorne fosfolipaze A2 (sPLA2) se vežejo na površino različnih membranskih struktur in cepijo sredinsko estrsko vez fosfolipidov, pri čemer sprostijo proste maščobne kisline (MK) in lizofosfolipide. Delovanje človeške sPLA2 iz skupine X (hGX) na celice raka dojke MDA-MB-231 vodi k povečanju tvorbe lipidnih kapljic (LK) in omogoči njihovo preživetje v stresnih pogojih z zmanjšano vsebnostjo hranil. Za učinke, ki jih ima hGX na celice MDA MB 231, je v veliki meri odgovorna predvsem oleinska kislina (OA), ki tako kot hGX povzroči nastanek LK in omogoči preživetje stradanih celic. MK, ki se sprostijo ob delovanju hGX, povzročijo spremembe v metabolizmu in tvorbi LK. LK rakavim celicam dajejo prednost v hipoksičnem in s hranili revnem tumorskem okolju, zagotavljajo vir energije ter ščitijo pred lipotoksičnim in oksidativnim stresom. Funkcija LK je neločljivo povezana s proteini, ki so prisotni na površini fosfolipidnega monosloja LK. Proteom LK se nenehno spreminja in je odvisen od razmer ter tipa celice. Dosedanje proteomske raziskave so pokazale, da proteom LK vsebuje 100–150 proteinov, za nekaj več kot 50 proteinov pa so prisotnost na LK tudi potrdili. Z LK povezani proteini spadajo v različne proteinske družine z raznolikimi funkcijami, ki so le redko povezane z lipidnim metabolizmom. V tem delu smo raziskali, kako delovanje hGX in enega izmed njenih produktov, OA, spremeni sestavo proteoma LK v celicah raka dojke. Ponovljivost in zanesljivost proteomskih rezultatov na izoliranih LK smo želeli izboljšati z izvedbo več tehničnih in bioloških ponovitev. Rezultate smo kvantificirali z uporabo dveh različnih pristopov kvantitativne proteomike (SILAC, angl. ''stable isotope labeling by amino acids in cell culture'' in LFQ, angl. ''label free quantification''). V izoliranih frakcijah LK smo zaznali 432 proteinov, vsaj 12 % (50/432) le-teh pa smo lahko uvrstili med proteine, ki so zelo verjetno povezani z LK. Med slednjimi je bilo 17 že znanih, "bona fide" proteinov LK. Velik del zaznanih proteinov (49 %, 213/432), za katere dejanske povezanosti z LK nismo mogli določiti, predstavlja potencialne nove kandidate proteinov LK. Preostali del zaznanih proteinov pa so zelo verjetno kontaminante iz drugih celičnih razdelkov (39 %, 169/432). Dobro poznani proteini LK so bili prisotni tudi pri celicah, ki niso bile izpostavljene povečani količini lipidov. Pri celicah, ki smo jih tretirali s hGX in OA, smo na LK zaznali proteine z različnimi funkcijami, med katerimi je največ proteinov lipidnega in oksidoredukcijskega metabolizma. LK celic, tretiranih z encimom hGX, so v primerjavi s celicami tretiranimi z OA in kontrolno skupino vsebovale večje število potrjenih proteinov LK. Dodatek OA ali hGX k celicam raka dojke je povzročil povečanje količine skoraj vseh potrjenih proteinov LK z izjemo perilipina 3. Trenda sprememb v količini proteinov sta podobna v celicah, tretiranih s hGX, in v tistih, tretiranih z OA. Raznolikost proteinov in spremembe v proteomu LK kažejo na pomembno vlogo LK v odzivu na stresni odgovor in homeostatske procese v celici.

Ključne besede

lipidi;lipidne kapljice;maščobne kisline;proteini;encimi;fosfolipaze;celice raka dojke;magistrska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [T. Nagode]
UDK: 577.15:577.115:618.19-006(043.2)
COBISS: 1538322627 Povezava se bo odprla v novem oknu
Št. ogledov: 682
Št. prenosov: 174
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Secreted phospolipase A2 changes proteom composition of lipid droplets in breast cancer cells
Sekundarni povzetek: Secreted phospholipase A2 (sPLA2) enzymes bind to the surface of various membrane structures and cleave the sn-2 ester bond of glycerophospholipids, releasing free fatty acids (FFAs) and lysophospholipids. The action of the human group X (hGX) sPLA2 on MDA-MB-231 breast cancer cells induces lipid droplet (LD) biogenesis and enables cell survival during starvation. FFAs, in particular oleic acid (OA), liberated from membrane phospholipids by the enzymatic activity of hGX sPLA2 are responsible for LD formation and the pro-survival effect of the enzyme. The released FFAs are incorporated into growing LDs, but also induce other changes in lipid metabolism and signalling that endow cancerous cells with a growth advantage in the hypoxic and nutrient-poor tumor microenvironment. LDs accumulate in stressed cancer cells, acting as sources of energy and providing protection against lipotoxic and oxidative stress. The functions of LDs are inherently determined by the proteins present on their surface, bound to the LD phospholipid monolayer. Dynamic alterations in the LD protein coat are crucial for the adaptation of LDs to fluctuating metabolic states. Currently, the high confidence lipid droplet proteome consists of 100–150 proteins in a prototypical mammalian cell, whereby the presence on LDs has been experimentally validated for more than 50 proteins. Interestingly, numerous proteins belonging to different functional groups, mostly not related to lipid metabolism, associate with LDs. Here we investigated the changes in the LD proteome of MDA-MB-231 breast cancer cells induced by treatments with hGX and one of its products, OA. In order to improve the reproducibility and reliability of our proteomic analyses of isolated LDs, we performed several technical and biological replicates. Changes in LD proteome were quantified with two different proteome profiling strategies (SILAC, "stable isotope labeling by amino acids in cell culture" and LFQ, "label free quantification"). In total, we identified 432 proteins in LDs isolated from MDA-MB-231 breast cancer cells. At least 12% (50/432) were classified as high confidence LD-associated proteins, including 17 already well recognized, "bona fide" LD proteins in mammalian cells. Based on the current literature, we could not assign a clear association with LDs for almost half of the detected proteins (49%, 213/432), suggesting that these could potentially represent new LD proteins. The rest of the detected proteins, however, were likely contaminants from other cell compartments (39%, 169/432). Interestingly, proteins involved in lipid and redox metabolism were the most abundant protein classes observed on LDs. Importantly, cells treated with hGX contained more high confidence LD proteins than untreated cells and those treated with OA. However, several well-known LD proteins were also detected in untreated cells that were not exposed to excess amounts of lipids. With the expection of perilipin 3, the addition of OA or hGX to breast cancer cells caused a significant increase in the amounts of high confidence LD proteins. A similar trend in the determined quantitative changes in the LD proteome were observed for cells treated with hGX and those with OA. The observed protein diversity and dynamics of the LD proteome suggest an involvement of LDs in homeostatic processes and the cancer cell stress response.
Sekundarne ključne besede: lipid droplets;phospholipase;fatty acids;breast cancer cells;lipids;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 1000377
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Strani: 56 str.
ID: 11210454