Identifikacija membranskih substratov cisteinskih katepsinov na celični liniji raka dojke SK-BR-3

Tršavec, Bine (avtor), Turk, Boris (mentor)

Cisteinski katepsini so proteaze, ki jih najdemo v lizosomih in sodelujejo pri znotrajcelični razgradnji proteinov. V patoloških pogojih, med katere spada tudi rak dojke, jih celice izločajo tudi v izvencelični prostor. Pomemben molekularni mehanizem, ki je povezan z razvojem raka je odcep ektodomene transmembranskih proteinov. Med proteaze, ki odcepljajo ektodomene, spadajo tudi nekateri predstavniki cisteinskih katepsinov. Za modeliranje delovanja proteaz in s tem podrobnejše razumevanje njihove vloge pri odcepu ektodomen na celični površini je potrebna identifikacija nabora substratov posameznih proteaz na različnih celičnih linijah. Celična linija raka dojke SK-BR-3 ima HER2 pozitiven fenotip, ki v povezavi z odcepom ektodomene s katepsini še ni bil raziskan. S pomočjo proteomske analize z masnim spektrometrom smo na površini celic omenjene celične linije identificirali 45 proteinskih substratov katepsinov K, L in S. Za primerjavo rezultatov smo naredili prenos western s protitelesi proti pleksinu B2, EGFR in HER2 na celičnih linijah MDA-MB-231 in SK-BR-3. Kljub različni stopnji izražanja HER2 pri omenjenih celičnih linijah, razlike pri odcepu ektodomene s prenosom western nismo uspeli potrditi. Vpliv obdelave s katepsini na nivoju celičnih funkcij smo analizirali s testoma migracije Transwell test in test s celjenjem rane na celičnih linijah MDA-MB-231 in SK-BR-3. Iz rezultatov migracijskih testov nismo mogli potrditi hipoteze o vplivu obdelave s katepsini na migracijo celic SK-BR-3.

• Jezik: Slovenski jezik
• Ključne besede: odcep ektodomene, katepsini, rak
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-116517
• COBISS.SI-ID: 17098755
• Datum objave v RUL: 26.05.2020

TRŠAVEC, Bine, 2020, Identifikacija membranskih substratov cisteinskih katepsinov na celični liniji raka dojke SK-BR-3 [na spletu]. Magistrsko delo. [Dostopano 18 junij 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=116517

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Identification of the membrane substrates of cysteine cathepsins in the breast cancer cell line SK-BR-3

Izvleček

Cysteine cathepsins are proteases found in lysosomes and play a crucial role in intercellular protein degradation. During pathological states such as breast cancer they are increasingly secreted into the extracellular space, where they can contribute to disease development. One of the main mechanisms during increased proteolytic activity in the extracellular space, which was also linked to cancer development, is ectodomain shedding. Among others, some proteases classified as cysteine cathepsins are known to be sheddases. For modelling the behaviour of proteases and thus determining their role in ectodomain shedding, it is crucial to identify all substrates of each sheddase at multiple different cell lines. Until now the protein substrates of cathepsins during ectodomain shedding at the cell surface of HER2-positive cell line SK-BR-3 have not yet been identified. Using proteomic screening of the shed peptides we identified 45 protein substrates of cathepsins K, L and S on the surface of the aforementioned cell line. Western blot analysis was performed against plexin B2, EGFR and HER2 both on SK-BR-3 and MDA-MB-231 cell lines to confirm the results of the proteomic analysis. The impact of ectodomain shedding using cathepsins on cell migration was tested with Boyden Transwell test and Wound healing test on cell lines MDA-MB-231 and SK-BR-3. However, the results of the migration assays did not allow us to draw a conclusion about the influence of cathepsin shedding on cell migration of the cell line SK-BR-3.

• Ključne besede: ectodomain shedding, cathepsins, cancer