Browsing by Subject "Mitochondrial Proteins"
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Item Characterization of a Small Molecule Smac Mimetic's Role in Inducing Apoptosis in Human Cancer Cells(2008-09-19) Yalcin-Chin, Asligul; Wang, XiaodongInhibitor of apoptosis proteins (IAPs) regulates apoptosis by inhibiting caspases. This inhibition mechanism is an escape from death used by some human cancers. Second mitochondria-derived activator of caspases (Smac), a mitochondria-released protein during apoptosis, binds to IAPs BIR domains with four amino acid residues (AVPI) and releases the inhibition caused on caspases by IAPs. With the idea of designing a Smac mimicking drug, that will induce apoptosis in cancer cells, we synthesized a small molecule Smac mimetic compound. I tested the ability of the Smac mimetic compound to induce apoptosis on several human cancer cells in combination with chemotherapeutic agents. Unexpectedly, in 25% of the cancer cells we tested, Smac mimetic treatment alone caused apoptosis. Of the cancer cells that were sensitive to Smac mimetic, MDA-MB231 human breast cancer cells and HCC44, HCC461, H2126 lung cancer cells had the highest sensitivity. In addition, a majority of the lung cancer cell lines I tested were sensitive to TNF and/or TRAIL in combination with Smac mimetic. We identified the target of Smac mimetic to be XIAP, cIAP1, and cIAP2 in both Smac mimetic induced and TNF/Smac mimetic induced apoptosis. Moreover, we were able to mimic the Smac mimetic effect by triple knockdown experiments of IAPs in TNF induced cell death. Furthermore, we identified the target of Smac mimetic to be XIAP in the TRAIL pathway. This work identifies the targets and mechanism of Smac mimetic induced cell death in cancer cells.Item Characterization of the Antiviral Effector IFI6(2018-11-26) Richardson, Ryan Blake; Yan, Nan; Schoggins, John W.; Levine, Beth; Pfeiffer, Julie K.The innate immune response is a critical line of host defense against invading pathogens. The production of interferon (IFN) and the subsequent expression of interferon stimulated genes (ISGs) are major contributors to the innate immune response, which establish an antiviral state in the cell. Flaviviruses such as dengue virus, Zika virus, and West Nile virus rely intimately on host pathways for completing a replication cycle, and have developed strategies to overcome the inhibitory effect of the innate immune response. To identify host factors required during an IFN response to flavivirus infection, a genome-wide CRISPR screen was carried out. Two of the top hits from the screen were IFI6, a previously identified ISG long predicted to be antiviral, and BiP, a luminal chaperone in the endoplasmic reticulum (ER). I questioned whether IFI6 was important for the antiviral response to flaviviruses and sought to investigate its role during infection. I confirmed the results from the CRISPR screen and showed that cells lacking IFI6 were insensitive to IFN, suggesting a key role in the innate immune response to flaviviruses. This was complemented by overexpression studies which showed IFI6 is potently inhibitory to flavivirus infection. I further demonstrated that BiP is required for an intact IFN response and importantly mediates expression of IFI6, which it binds in a chaperone-dependent manner. I also showed that IFI6 is localized to the ER and is an integral membrane protein. Importantly, IFI6 acts during the flavivirus life cycle to inhibit replication and formation of replication complexes, which are formed by rearrangement of ER membranes. IFI6 specifically inhibits flaviviruses, since other viruses that replicate at the ER such as hepatitis C virus (HCV) are not affected by IFI6. I hypothesize the key to this specificity lies in the orientation of the replication complexes - HCV complexes extend outwards into the cytoplasm while flaviviruses bud inwards into the lumen. Taken together, these data support a model where IFI6 is sensitive to membrane alterations specifically induced by flaviviruses but not other viruses, which provides the innate immune response with a potent and specific ISG to block viral infection.Item In Vivo Studies of Yeast Mitochondrial Intron Splicing : Ectopic Branching and a Screen for Nuclear Encoded Splicing Factors(2006-08-11) Nyberg, Tarah Michelle; Perlman, Philip S.The splicing mechanism of group II introns is analogous to that of nuclear introns and it is generally thought that both share a common ancestor. This work contains two studies of group II intron splicing in yeast mitochondria. Previous studies done in collaboration with Dr. Anna Pyle at Yale identified several important determinants for in vitro branch-site selection of intron aI5gamma : the presence of a bulged A(A880), the 5' flanking GU base pair and the branch location within domain VI. I confirmed the in vitro findings in vivo and show that displacing the branch adenosine by one nucleotide in either direction can support branching at the shifted bulged A in vivo. Returning the base-pairs flanking the shifted branch-points to GU pairs increased both the efficiency and fidelity of branching at the ectopic branch A. However, for the shifted down ectopic branch A, it is not the presence of the GU pair flanking the branch that restores branching but the presence of a GC pair located two base-pairs above the branch. This finding is consistent with our observations that for the wild-type branch location, the branch environment above and below the branch are distinct. It appears that the short stem below the branch is important for the second splicing step. The goal of the second project was to identify novel nuclear genes that are involved in mitochondrial intron splicing. Based on the yeast genome project and several recent proteomic studies of yeast mitochondria, we identified 808 nuclear genes coding for potential mitochondrial proteins that can be deleted without lethality. Of these, 476 deletion strains retain a complete copy of the mtDNA (13 introns) and have a respiratory growth defect. Those strains were screened by northern blot analysis for intron splicing defects. I observed the expected splicing defects in strains deleted for MSS18, CBP2 and PET54. I observed a novel splicing pattern in strains deleted for IMP1, CBS2, PET111, MNE1, AAT1, ATP10 and PIF1.