Browsing by Subject "Apoptosis"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    The design and synthesis of constrained AVPI peptidomimetics
    (2008-12) Kending, Cory
    Recent studies have demonstrated that breast cancer cell lines are sensitive to the XIAP (X-linked inhibitor of apoptotic proteins) antagonist Smac (second mitochondrial activator of caspases). Specifically the terminal tetrapeptide, AVPI, inhibits XIAP and thus sensitizes the breast cancer cells to various apoptotic therapeutics. These peptides are poor drug candidates due to their size, hydrophilicity and potential for peptidase cleavage. To address these issues we systematically synthesized peptide mimics that are constrained at various torsion angles along the peptide backbone. The compounds were tested in vivo and in vitro utilizing the laboratories of the Mayo Medical School. One compound, HCl*NH2-(R)-αMe-γ-Lactam-L-Val-L-Pro-L-Ile-OMe (3.1), increased the apoptotic signals above the assay background. Retro-modeling analysis utilizing the Schödinger flexible docking program suite did not provide an easy explanation for why this might be the case. The in silico models suggest that compound 3.1 interacts with only the primary pocket of the AVPI binding site and does not interact, like the native peptide, with both pockets in the active site. The R stereochemistry of the bicyclic compounds is predicted by our models to have a binding efficacy above that of the native peptide presumably due to the interaction with both pockets of the binding site as well as d-orbital interactions of the thiazole ring system with the neighboring tryptophan. The S stereochemistry was also initially proposed as a negative control for the biological assays, however, the instability of the molecule provided several daunting challenges to an already challenging and low yielding synthesis and the negative controls were abandoned when it was clear that the synthesis was futile. Our research shows limited evidence that our AVPI peptidomimetics are a potential scaffold to base other drug-like molecules upon. Future biological data from the bicyclic compounds will hopefully prove our hypothesis and molecular modeling studies correct.
  • Thumbnail Image
    Item
    Functional characterization of the candidate colorectal cancer gene CNOT1
    (2013-12) Zhao, Lei
    A Sleeping Beauty transposon-mediated forward genetic screen in mice identified Cnot1 as a candidate colorectal cancer gene (CRC). CNOT1 is a central subunit of the CCR4-NOT (CNOT) deadenylase complex that regulates mRNA expression through multiple mechanisms, such as deadenylation. Our study suggests a model that CNOT1 protects cells from severe endoplasmic reticulum (ER) stress induced apoptosis through limiting the levels of TXNIP via promoting deadenylation and degradation of TXNIP mRNA transcripts. We also fund that CNOT1 plays a role in limiting cellular ER stress. Given that TXNIP is activated to promote apoptosis in response to extreme cellular stresses, such as ER stress, and that cancer cells commonly experience high levels of ER stress, increased activity of CNOT1 may be a mechanism that allows cancer cells to survive by protecting against cell stress-induced apoptosis.
  • Thumbnail Image
    Item
    Regulation of stress associated protein kinase signaling pathways and apoptosis by herpes simplex virus Type 1 infected cell protein 27.
    (2010-02) Gillis, Peter A.
    Infected cell protein 27 (ICP27) is an immediate early (IE) herpes simplex virus type 1 (HSV-1) regulatory protein. During the course of a HSV-1 infection, ICP27 has been implicated in a variety of functions important for viral replication including host shut-off, viral gene expression, stress activated protein kinase (SAPK) activation, and apoptosis inhibition. However, many of these studies have relied on infections with viral deletion mutants to make these observations, but have failed to separate the direct and indirect effects of ICP27 on viral replication and cellular function. To examine the direct functions of ICP27, we engineered stable HeLa cell lines that inducibly express ICP27. We showed that ICP27 expression is sufficient to activate p38 signaling to a level similar to that which is observed during wild type HSV-1 infection. Interestingly, ICP27 expression is only able to induce a modest level of JNK signaling, compared to the high levels observed during WT HSV-1 infection. Using chemical inhibitors, we show that the ICP27-mediated activation of p38 signaling triggers apoptosis in the inducible cell lines demonstrating that ICP27 is a pro-apoptotic factor. However, it is likely that additional downstream ICP27-dependent viral factors inhibit apoptosis during HSV-1 infection. In an effort to better understand the regulation of apoptosis ICP27, we wanted to determine the effects of ICP27 expression on NF-B activation. During HSV-1 infection ICP27-mediated JNK signaling is required for the activation of NF-B, which is required for the inhibition of apoptosis. Using our inducible cell lines, we found that ICP27 expression alone is unable to activate NF-B, suggesting that at least one additional ICP27-dependent viral factor is required. We demonstrate that the HSV-1 protein UL37 is expressed in an ICP27/JNK signaling-dependent manner and is capable of activating NF-B. Furthermore, expression of UL37 in an ICP27-independent fashion is able to delay apoptosis induced by infection with an ICP27 null mutant. Finally, we wanted to characterize the effects of ICP27-mediated p38 signaling on normal cellular function. Using the inducible cell lines, we demonstrate that ICP27 and subsequent p38 signaling are capable of stabilizing the ARE-containing IEX-1 transcript in the absence of infection, by manipulating cellular pathways regulating mRNA decay. During the course of these studies, we also observed that ICP27 expression activates elements of the cellular integrated stress response and the endoplasmic reticulum stress pathways leading to eIF2α phosphorylation and IRE1 activation, respectively. The cellular response to ER stress induced by ICP27 could provide a mechanism for induction of SAPK signaling. These combined results indicate that ICP27 functions as an important regulator of cell fate during HSV-1 infection.