Browsing by Subject "Proteolytic enzymes"

Now showing 1 - 4 of 4
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    Identification of the Pba1 and Pba2 Binding Sites on 20S Core Particle Intermediates
    (2013-07-12) Hammack, Lindsay Jo; Kusmierczyk, Andrew; Malkova, Anna; Randall, Stephen Karl, 1953-; Atkinson, Simon
    The proteasome is responsible for breaking down the majority of the proteins in the cell. However, a complete understanding of how this large multi-subunit protease is assembled is currently lacking. Proper and timely assembly of the proteasome is critical for the functioning of the ubiquitin-proteasome pathway, defects in which have been associated with several different cancers. A recently discovered heterodimeric proteasome assembly chaperone, Pba1p-Pba2p, has been suggested to prevent the assembly process from straying off path. Pba1p-Pba2p associates with proteasomal assembly intermediates via C-terminal HbYX motifs. The HbYX motif is a tri-peptide sequence containing a hydrophobic residue (Hb) followed by a tyrosine (Y), then any amino acid (X). This motif was originally identified in proteasomal activators, and shown to mediate the association of activators with the proteasome by inserting into intersubunit pockets on either end of the proteasome. There are seven unique intersubunit binding pockets, located between neighboring α subunits on the proteasome, to which a HbYX-containing protein can bind; which of these pockets Pba1p-Pba2p binds to remains elusive. I attempted to identify where Pba1p and Pba2p bind via a crosslinking approach. Specific residues were mutagenized to cysteines on Pba1p, Pba2p, and the individual α subunits in order to generate crosslinkable species. By exposing yeast cells expressing these crosslinkable proteins to mild oxidizing conditions, I attempted to trap the Pba1p and Pba2p α intersubunit pocket interactions. In order to optimize crosslinking conditions, the assay was modified several ways. Additionally, measures were taken to increase detection of the crosslinked species via immunoblotting. Despite the efforts to improve the crosslinking and detection, I was unable to successfully detect a crosslinked species. However, crosslinking is a reasonable method to identify the Pba1p and Pba2p proteasomal binding sites, having been successfully used to identify binding sites for other HbYX-motif-containing proteins; further assay optimization should yield Pba1p and Pba2p proteasomal crosslinks.
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    mTORC1 contributes to ER stress induced cell death
    (2012-12) Babcock, Justin Thomas; Quilliam, Lawrence; Atkinson, Simon; Nakshatri, Harikrishna; Wek, Ronald C.
    Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.
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    Tumor-stroma interaction mediated by tissue transglutaminase in pancreatic cancer
    (2015-08) Lee, Jiyoon; Matei, Daniela Elena; Harrington, Maureen A.; Herbert, Brittney-Shea; Xie, Jingwu
    Pancreatic ductal adenocarcinoma (PDA) is a deadly disease due to early metastasis and resistance to chemotherapy. PDA is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is abundantly expressed in pancreatic cancer cells and crosslinks proteins through acyl-transfer transamidation between glutamine and lysine residues. The objective of the study was to determine the functions of TG2 in the pancreatic stroma. Orthotopic pancreatic xenografts and co-culture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. We showed that TG2 secreted by cancer cells is enzymatically active and renders the stroma denser by crosslinking collagen, which in turn activates fibroblasts and stimulates their proliferation. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are transcription factors involved in mechanotransduction. The TG2-mediated fibrosis-rich, stiff microenvironment conveys mechanical cues to cancer cells leading to activation of YAP and TAZ, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells led to decreased size of pancreatic xenografts and increased sensitivity of xenografts to gemcitabine. Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the crosstalk between cancer cells and the stroma, fundamentally impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.