No description available.

The ubiquitin-proteasome system is an important proteolytic pathway implicated in the formation of neurofibrillary tangles from hyperphosphorylated tau, a pathological hallmark of tauopathies such as Alzheimer’s disease. We hypothesize that proteasome inhibition will result in tau accumulation, leading to the formation of aggregates. We established a combined pharmacological and genetic model of proteasome inhibition in SHSY-5Y neuroblastoma cells. Through lentiviral infection, we expressed a mutant T1A form of the β5 proteasome subunit to impair the chymotryptic proteolytic activity of the proteasome. Cells were then treated with different pharmacological inhibitors of the proteasome to further exacerbate the effects of mutation-mediated proteasome inhibition and tested for changes in tau expression. Contrary to our hypothesis, chronic proteasome inhibition and exposure to mild oxidative stress concurrently resulted in increased tau degradation. Therefore, although chronic proteasome inhibition was insufficient to induce changes in tau turnover, it rendered cells more vulnerable to oxidative insult.

The ubiquitin-proteasome system is an important proteolytic pathway implicated in the formation of neurofibrillary tangles from hyperphosphorylated tau, a pathological hallmark of tauopathies such as Alzheimer’s disease. We hypothesize that proteasome inhibition will result in tau accumulation, leading to the formation of aggregates. We established a combined pharmacological and genetic model of proteasome inhibition in SHSY-5Y neuroblastoma cells. Through lentiviral infection, we expressed a mutant T1A form of the β5 proteasome subunit to impair the chymotryptic proteolytic activity of the proteasome. Cells were then treated with different pharmacological inhibitors of the proteasome to further exacerbate the effects of mutation-mediated proteasome inhibition and tested for changes in tau expression. Contrary to our hypothesis, chronic proteasome inhibition and exposure to mild oxidative stress concurrently resulted in increased tau degradation. Therefore, although chronic proteasome inhibition was insufficient to induce changes in tau turnover, it rendered cells more vulnerable to oxidative insult.

No description available.

Three of the best-characterized prions of Saccharomyces cerevisiae are [PSI+], [URE3], and [PIN+]. This study focuses on the prions [PSI+] and [PIN+]. [PSI+] is the prion isoforms of the protein Sup35 that functions as a eRF3 translational termination factor. The presence of [PSI+] is detected by the partial loss of function of Sup35. The prion [PIN+] is the isoform of the protein Rnq1, and this proteins function is unknown. The presence of the prion [PIN+] is necessary for the de novo formation of the prion [PSI+] (Derkatch et al., 1997). The chaperone, Hsp104, belongs to an evolutionary conserved Hsp100 family of proteins that participate in a various number of cellular processes (Schirmer et al., 1996). Hsp104, in particular, is responsible for the cells adaptation to heat shock, it controls spore viability and the long-term viability of starving vegetative cells. (Sanchez and Linquist, 1990; Sanchez et al.,1992) It is an ATPase that has been shown to promote solubilization of aggregated protein (Parsel et al., 1991). A unique relationship exists between Hsp104 levels within the cell and the maintenance of the prion [PSI+]. The over production of Hsp104 eliminates [PSI+] (Chernoff 1995). This seems logical considering Hsp104 is a disaggregase, and it is reasonable to assume that the over production provides sufficient resources to break the aggregates into portions that are accessible to either other chaperones which would facilitate the proper folding or perhaps the system responsible for the elimination of unusable proteins, such as the ubiquitin-proteasome system. This study examines the role of the ubiquitin-proteasome system in curing of [PSI+] by Hsp104. The role of alternate pathways, in which the prion isoform is refolded into it correct, functional conformation by the action of the chaperones Ssb1 and Ssb2 is examined. These results suggest that the combination of both the degradation pathway and the refolding of proteins are involved in curing of [PSI+] by Hsp104 over production.

The ubiquitin-proteasome system regulates protein degradation. Although proteasomes localize in the nucleus of proliferating Saccharomyces cerevisiae, they are sequestered into cytoplasmic proteasome storage granules (PSG) in quiescence. Although important for cell cycle regulation and mediating external stressors, the content and structure of these membraneless PSGs remain unknown. Yeast deletion genetic screens identified several ubiquitin-related genes involved in proteasome sequestration into PSGs. This study aims to determine whether changes in free ubiquitin levels or ubiquitin post-translational modifications affect proteasome dynamics in quiescence. Unlike the wild-type, PSGs were not seen in catalytically inactive Ubp6 mutant strains in quiescence and proteasomes failed to be imported into the nucleus upon the resumption of cell growth. Although no significant differences in proteasome configurations were observed, Western blot analysis of these mutants suggests the presence of post-translationally modified monoubiquitinated proteasomes in quiescence. Ubiquitin modification may target proteasomes towards lysozomal degradation rather than into PSGs for storage.

Since Bortezomib®, a proteasome inhibitor, was approved by US FDA for the treatment of multiple myeloma in 2003, proteasome is recognized as one of the most promising targets for cancer therapeutics. The proteasomes play a critical role in regulating the level of cellular proteins and recycling damaged and misfolded proteins. Although the activity of the proteasome is essential for normal cells, it is especially critical for the proliferation and survival of cancer cells. In an attempt to develop effective and safe proteasome inhibitor-based anticancer drugs, the Lee laboratory created a chemical library by a hybrid approach using a 4-piperazinylquinoline scaffold and a sulfonyl phamarcophore. It is known that the chloroquine scaffold possesses a weak proteasome inhibition activity, and chloroquine itself preferentially kills malignant cells over non-cancer cells, alone or in combination with other therapeutics. To identify compounds with desirable anticancer activities, I have screened the aforementioned chemical library. The screening yielded several hits with substantial efficacy and selectivity against malignant cells. In this thesis, I describe the functional mechanism of VR23, one of the most promising compounds identified from my screening, as it kills cancer cells up to 17 fold more effectively than non-cancer cells. Molecular docking and substrate competition studies revealed that VR23 binds to the β2 peptide of the 20S proteasome catalytic subunit. The IC50 value of VR23 in inhibiting trypsin-like proteasome activity is 1.0 nM. VR23 is also substantially effective in inhibiting chymotrypsin-like proteasome activity (IC50, 50-100 nM). The inhibition of proteasome activity by VR23 led to the accumulation of ubiquitinated cyclin E at centrosomes. This, in turn, induces abnormal centrosome amplification by a de novo centrosome synthesis pathway in cancer cells, but not in non-cancer cells. The presence of multiple centrosomes in single cancer cells results in cell cycle arrest at prometaphase and, eventually, cell death by apoptosis. Thus, VR23 possesses a very desirable property as a safe anticancer drug.

Indiana University-Purdue University Indianapolis (IUPUI) / The 20S proteasome, a multi-subunit protease complex, present in all domains of life and some orders of bacteria, is involved in degradation of the majority of cellular proteins. Structurally, it is made of α and β subunits arranged in four heptameric rings, with inner two β-rings sandwiched between outer two α-rings. The 20S proteasome in prokaryotes usually has one type of α and one type of β subunits, whereas eukaryotes have seven distinct types of α and seven distinct types of β subunits. Unlike the highly conserved structure of proteasome, its assembly pathway is different across the domains. In archaea and eukaryotes, proteasome assembly begins with α subunit interactions leading to the α-ring formation. By contrast, bacterial proteasome assembly pathway bypasses the α-ring formation step by initiating assembly through an α and β subunit interaction first. These early interactions are not well understood due to their highly rapid and dynamic nature. This dissertation focused on understanding the early events in proteasome assembly and contributed three significant findings. First, the archaeal proteasome assembly can also begin without formation of α-rings, demonstrating the coexistence of a bacterial-like assembly pathway. Second, a novel assembly intermediate was identified in yeast, and its composition argues for the presence of a similar α-ring independent assembly pathway. Third, the assembly chaperone Pba3-Pba4 prevents the formation of high molecular weight complexes arising from spontaneous and non-productive interactions among the α subunits. These findings provide a broader understanding of proteasome biogenesis and suggest considering proteasome assembly event as a network of interactions rather than a linear pathway. The results also shed light on assembly chaperone’s contribution in increasing the efficiency of proteasome assembly by streamlining the productive interactions. / 2020-12-07

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.