Bag-1L'in ubikuitin/proteazom sisteminde rolünün meme kanserinde incelenmesi

An anti-apoptotic protein BAG-1, with its adaptor function, can interact with a diverse array of molecular targets such as Hsp70/Hsc70 chaperones, proteasome machinery, Bcl-2, Raf-1 kinase, nuclear hormone receptors and DNA, and is found to involve in the regulation of a wide variety of cellular processes. In human cells, three functionally distinct and differentially localized BAG-1 isoforms originating from a single mRNA are expressed through alternative translation and these three isoforms include BAG-1S (33 kDa), BAG-1M (46 kDa) and BAG-1L (52 kDa), which have distinct N-terminus. BAG-1L, which possess a nuclear localization signal is a largely nuclear protein, whereas BAG-1M partitions between nucleus and cytoplasm. BAG-1 with its isoforms is a candidate protein to form hubs to transmit signals for survival. It has been reported that relatively high BAG-1L levels are observed in tumor cells. Protein folding process occurs in the endoplasmic reticulum. This folding process involves heat shock proteins, lectin proteins, disulfide isomerases present in the ER. In protein folding, N-glycosylation occurs which increases protein stability. N-linked glycans increase protein solubility and directly affect protein folding pathways. Calnexin (ER membrane bound) and Calreticulin (ER lumen) are lectin chaperones and support protein folding. Lectin chaperones connect to the last glucose and then control the folding process. In addition, lectin chaperones interact non-covalently with oxidoreductase PDI (ERp57), thus triggering disulfide bond formation and isomerization on the substrate. PDI is a member of the disulfide isomerase protein family and is found in the ER lumen. The formation of disulfide bonds limits the conformational flexibility of the proteins and increases the structural stiffness of the proteins. Despite the quality control mechanism in ER, when protein folds are inadequate and the protein can not fold properly, aberrant proteins accumulate in the ER. Therefore, the activation of the unfolded protein response (UPR) occurs. Through ERAD pathway, which is part of the UPR system, abnormal protein retrotranslation from the ER to the cytosol occurs. The ERAD pathway consists of a series of processes that mediate the retrotranslocation of selected aberrant proteins and the ubiquitin-dependent proteasomal degradation. The energy that drives substrate dislocation is derived from adenosine triphosphate (ATP) hydrolysis by the abundant, ubiquitin-dependent hexameric AAA+ ATPase VCP (p97). VCP is recruited to ERAD dislocation complexes through direct interactions with several ERAD components. ERAD substrate polyubiquitination functions as a binding site that facilitates extraction by VCP and acts as the signal for proteasomal degradation. The addition of ubiquitin occurs via sequential enzymatic reactions involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase that mediates the final transfer of ubiquitin to the substrate. CHIP (E3 ligase) is part of a system that directs misfolded proteins from chaperones to proteasomes. Two modulators, BAG-1 and CHIP, have implicated in cytosolic degradation pathways, xxii putting molecular chaperones at the center of the decision process between folding and degradation of non-native polypeptides. The ubiquitin/proteasome system, the interaction partner of BAG-1, degrades misfolded or unfolded proteins. Investigating the effect of BAG-1 on the estrogen receptor has found that BAG-1L promotes estrogen-dependent transcription but BAG-1M or BAG-1S do not increase transcription of ER. BAG-1L interacts with estrogen receptors and enhances receptors activity. Studies on proteasome have shown that toxic effects of proteasome inhibition in cancer cells are more sensitive than normal cells. Although the mechanism behind the higher susceptibility of cancer cells is not known, there is a high probability of using the proteasome to regulate proliferation and anti-apoptotic pathways. BAG-1, an adaptor protein in the ubiquitin/proteasome system, can be used as a drug target in cancer due to cell survival effect. Here, we adopted a proteome-based strategy comprising isolation of BAG-1L associated complexes from breast cancer and epithelial cell lines as well as breast tissues. Identification of the proteins involved in BAG-1L associated complexes to ultimately delineate the adaptor role of BAG-1L in breast cancer. Proteins involved in BAG-1L associated complexes were identified via LC-MS/MS analysis with an BAG-1L isoform-specific manner. Further analyses with bioinformatics tools hinted the involvement of BAG-1L isoform in protein quality control system, which includes the action of endoplasmic reticulum-associated degradation (ERAD) mechanism to maintain intracellular homeostasis of cellular proteins. Interaction of BAG-1L isoform with the proteasome were poorly characterized, detailed understanding of the involvement of BAG-1L isoforms in the ubiquitin mediated proteasomal degradation is lacking. Determined interactions which includes specifically VCP/p97 and Rad23, components of proteasome machinery, set a critical role for BAG-1 in the ubiquitin-dependent proteasomal degradation.