Institutional creator:

Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN

Contributor:

Nałęcz, Katarzyna Anna (1952- ) : Supervisor

Publisher:

Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN

Place of publishing:

Warszawa

Description:

141 pages : illustrations ; 30 cm ; Bibliography ; Summary in English

Degree name:

PhD in Biological Sciences

Degree discipline :

biological sciences

Degree grantor:

Nencki Institute of Experimental Biology PAS ; degree obtained in 2024

Type of object:

Thesis

Abstract:

ATB0,+ is a membrane amino acid transporter, encoded by the SLC6A14 gene, with broad substrate specificity, transporting all neutral (index „0”) and basic (index „+”) amino acids in a sodium and chloride dependent way. ATB0,+ (SLC6A14) level is upregulated in many cancers, including breast cancer, in which its activity is important for growth and unlimited proliferation of transformed cells. SLC6A14 is an ideal candidate for targeting cancer therapies, as it also transports many drug precursors and amino acid-derived drugs such as valacyclovir and valgancyclovir. Previous analysis of the SLC6A14 proteome detected, among others, several heat shock proteins, including HSP70 (HSPA14) and HSP90-beta. This dissertation focuses on verifying the interaction between transporter and HSP proteins in the process of SLC6A14 exit from the endoplasmic reticulum. Interaction of the overexpressed transporter with heat shock proteins was confirmed in immunoprecipitation and immunofluorescence experiments, while direct interaction was shown in proximity ligation assay. Cell surface localization of the transporter was confirmed using biotinylation of cell surface proteins. Treatment with HSP90 and HSP70 inhibitors - radicicol or VER155008 respectively resulted in a dramatic decrease of SLC6A14 presence in the plasma membrane, an effect of the total transporter diminution. Possibility of interaction between SLC6A14 and HSP90 was tested by measuring ATPase activity of purified, recombinant HSP90-beta protein in an in vitro system. HSP activity was measured in the presence of particular peptides, representing fragments selected from the N- and C-terminus of the transporter, and it was shown that only two peptides, corresponding to C-terminal fragments located closer to the 12th transmembrane domain of SLC6A14, affected the activity although in opposite manner. The results indicate that the C- terminal fragment, which is essential for the exit of SLC6A14 from the endoplasmic reticulum via SEC24C binding, is also involved in altering the activity of HSP90-beta, suggesting that the same transporter fragment binds both HSP90-beta and SEC24C and is responsible for the proper folding of SLC6A14 and its further transport to the plasma membrane. Since high expression of both SLC6A14 and HSP90-beta has been observed in various types of cancer, we also decided to test whether HSP90-beta could act as a regulator of endogenous SLC6A14 activity in breast cancer cell lines. The study was performed on estrogen receptor-positive breast cancer cell lines and a control epithelial one. The direct interaction between SLC6A14 and HSP90-beta was confirmed by proximity ligation assay in all selected lines, but at a significantly higher level in the cancer lines, when compared to the control. The uptake of radioactive leucine, a high-affinity substrate of SLC6A14, was significantly reduced after treatment with the HSP90-beta inhibitor, in breast cancer cells with estrogen receptor (ER-α+) present. In the non-tumorigenic MCF 10A cell line, there was no effect of the HSP90 inhibitor on leucine accumulation, in the presence of chlorine ions, and leucine accumulation was much lower than in breast cancer lines. However, radicicol decreased interaction between SLC6A14 and HSP90-beta. This suggest that SLC6A14 synthesis is lower in non-tumorigenic cells, being much higher in cancer cells and this points to the involvement of HSP90-beta in regulation of SLC6A14 activity. Moreover, a cell viability assay revealed increased cell death after treatment with inhibitors: α-methyltryptophan (SLC6A14) and radicicol (HSP90), when given separately and the combination of these inhibitors significantly increased this effect. The observation that α-methyltryptophan and radicicol at low concentrations cause a synergistic cytotoxic effect in malignant breast cancer cell lines appears to be a potential strategy for combination therapy.

Detailed Resource Type:

PhD Dissertations

Resource Identifier:

oai:rcin.org.pl:243071

Source:

IBD PAN, call no. 20573

Language:

pol

Language of abstract:

eng

Rights:

Rights Reserved - Free Access

Terms of use:

Copyright-protected material. May be used within the limits of statutory user freedoms

Copyright holder:

Publication made available with the written permission of the author

Digitizing institution:

Nencki Institute of Experimental Biology of the Polish Academy of Sciences

Original in:

Library of the Nencki Institute of Experimental Biology PAS

Access:

Open