Matching Items (3)
Filtering by
- Creators: Jacobs, Bertram
Description
Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background.
ContributorsLeiser, Owen Paul (Author) / Misra, Rajeev (Thesis advisor) / Jacobs, Bertram (Committee member) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2010
Description
Programmed cell death plays an important role in a variety of processes that promote the survival of the host organism. Necroptosis, a form of programmed cell death, occurs through a signaling pathway involving the protein kinase RIPK3. In response to vaccinia virus infection, necroptosis acts through RIPK3 and the adaptor protein DAI to inhibit further viral replication in host cells. Stress granules are accumulations of mRNAs that have stalled in translation due to cellular stress. The toxin arsenite is a canonical inducer of stress granule formation and can cause necroptosis. By initiating necroptosis with arsenite and vaccinia virus, this research project investigated the roles of necroptosis proteins and their localizations into stress granules. The two aims of this research project were to determine if stress granules are important for arsenite-induced necroptosis, and whether the proteins DAI, RIPK3, MLKL, and G3BP localize into stress granules. The first aim was investigated by establishing a DAI and RIPK3 expression system in U2OS cells; arsenite was then used to treat the U2OS cells as well as U2OSΔG3BP cells, which are not able to form stress granules. The second aim was carried out by designing fluorescent tagging for the necroptosis proteins in order to visualize protein localization with fluorescent microscopy. The results showed that arsenite induces DAI-dependent necroptosis in U2OS cells and that this arsenite-induced necroptosis requires stress granules. In addition, it was determined that vaccinia virus induces DAI-dependent necroptosis that also requires stress granules. This project contributes to a greater understanding of the roles of DAI and RIPK3 in necroptosis, as well as the roles of stress granules in necroptosis, both of which are important in research regarding viral infection and cellular stress.
ContributorsGogerty, Carolina (Author) / Jacobs, Bertram (Thesis director) / Langland, Jeffrey (Committee member) / Jentarra, Garilyn (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Music (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Programmed cell death plays an important role in a variety of processes that promote the survival of the host organism. Necroptosis, a form of programmed cell death, occurs through a signaling pathway involving receptor-interacting serine-threonine protein kinase 3 (RIPK3). In response to vaccinia virus infection, necroptosis is induced through DNA-induced activator of interferon (DAI), which activates RIPK3, leading to death of the cell and thereby inhibiting further viral replication in host cells. DAI also localizes into stress granules, accumulations of mRNAs that have stalled in translation due to cellular stress. The toxin arsenite, a canonical inducer of stress granule formation, was used in this project to study necroptosis. By initiating necroptosis with arsenite and vaccinia virus, this research project investigated the roles of necroptosis proteins and their potential localization into stress granules. The two aims of this research project were to determine whether stress granules are important for arsenite- and virus-induced necroptosis, and whether the proteins DAI and RIPK3 localize into stress granules. The first aim was investigated by establishing a DAI and RIPK3 expression system in U2OS cells; arsenite treatment or vaccinia virus infection was then performed on the U2OS cells as well as on U2OSΔΔG3BP1/2 cells, which are not able to form stress granules. The second aim was carried out by designing fluorescent tagging for the necroptosis proteins in order to visualize protein localization with fluorescent microscopy. The results show that arsenite induces DAI-dependent necroptosis in U2OS cells and that this arsenite-induced necroptosis likely requires stress granules. In addition, the results show that vaccinia virus induces DAI-dependent necroptosis that also likely requires stress granules in U2OS cells. Furthermore, a fluorescent RIPK3 construct was created that will allowfor future studies on protein localization during necroptosis and can be used to answer questions regarding localization of necroptosis proteins into stress granules. This project therefore contributes to a greater understanding of the roles of DAI and RIPK3 in necroptosis, as well as the roles of stress granules in necroptosis, both of which are important in research regarding viral infection and cellular stress.
ContributorsGogerty, Carolina (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Jentarra, Garilyn (Committee member) / Arizona State University (Publisher)
Created2021