Herpes simplex virus entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in herpes simplex virus entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on herpes simplex virus capsid transport. Herpes simplex virus can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, herpes simplex virus successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. Herpes simplex virus immediate-early protein ICP0 is a multifunctional regulator of herpes simplex virus infection. Late in infection ICP0 interacts dynamically with cellular proteasomes. ICP0 has a RING finger domain with E3 ubiquitin ligase activity that is necessary for its IE functions. The fundamental and functional properties of ICP0 that is present in the virion tegument layer have not been well characterized. For these reasons, I sought to characterize tegument ICP0 and determine the role of tegument ICP0 during proteasome-dependent entry of herpes simplex virus. Protein compositions of wild-type and ICP0 null virions were similar, suggesting that the absence of ICP0 does not grossly impair virion assembly. Virions with mutations in the RING finger domain contained greatly reduced levels of tegument ICP0, suggesting that the domain influences the incorporation of ICP0. Virion ICP0 was resistant to removal by detergent and salt and was associated with capsids, features common to inner tegument proteins. ICP0 mutations that resulted in the absence of ICP0 in the tegument layer, allow herpes simplex virus to enter cells independently of the proteasome activity. I propose that proteasomal degradation of virion and/or host proteins is regulated by ICP0 to allow for efficient delivery of incoming herpes simplex virus capsids to the nucleus.

Thesis advisor: Marc Muskavitch / The ubiquitin-proteasome system (UPS), composed of classes of proteins central to the process of cellular protein turnover in eukaryotes, is essential to the life cycle of the malaria parasite, Plasmodium falciparum. Although the UPS has been well characterized in other organisms, the extent of its involvement in different stages of P. falciparum growth and development has not been investigated in depth. MG132, a small-molecule proteasome inhibitor known to target the 20S proteasome core (part of the catalytic center for selective protein degradation), has been used successfully in many research studies that require proteasome inhibition. We present data supportive of the conclusion that MG132 is highly effective as a tool for P. falciparum research. In this thesis, I describe the effects of partial and complete proteasome inhibition on parasite growth and development by the use of variable concentrations of MG132. I also assess the effects of MG132 on 20S P. falciparum proteasome enzymatic activities. I have generated parasite lines that exhibit tolerance, or low-level resistance, to MG132, through intermittent compound exposure. Sequencing of the catalytic β-5 subunit of the MG132-tolerant parasites reveals non-synonymous point mutations in three tolerant parasite lines. The use of MG132 as a tool compound for study of the UPS in P. falciparum facilitates research into detailed roles of the proteasome using reversible partial and complete inhibition. MG132-tolerant lines are also valuable tools for studying the genesis of different levels of drug resistance and cross-resistance in parasite evolution. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Neste trabalho serão apresentados resultados sobre três sistemas proteícos diferentes: Lisozima, Crioglobulina e Proteassomo, analisados a partir, principalmente, da técnica de espalhamento de Raios X a baixos ângulos. Técnicas como microscopia eletrônica de transmissão e espalhamento de luz dinâmico foram utilizadas como técnicas complementares. Os resultados apresentados demonstram o potencial único que a técnica de espalhamento a baixos ângulos possui no estudo de sistemas em solução. Os dados correspondem a um estudo em um grande intervalo de tamanhos para as proteínas estudadas (15kDa to 750 kDa), requerendo diferentes abordagens em cada caso. Como será mostrado, para casos onde se tem um meio monodisperso, diversas metodologias de modelagem podem ser utilizadas. Para o sistema composto de Lisozima, por se tratar de uma proteína amplamente estudada na literatura, é interesante poder comparar os resultados encontrados com os já publicados e observar sua estabilidade em solução. O peso molecular calculado a partir dos dados de espalhamento foi 15kDa, que está em bom acordo com a esperado para esta proteina (14,6 kDa). Além disso, ao variar-se a concentração da proteína em solução, é possível observar um fator de interação entre as partículas para maiores concentrações. Esse fator de interação pode ser considerado próximo ao de esferas rígidas. Para o sistema de Crioglobulinas, houve uma dificuldade na purificação da amostra, mas ainda assim apresentaremos alguns resultados interessantes, em que a técnica de SAXS fornece informações sobre a flexibilidade de proteínas e as análises para as amostras de um pool de imunoglobulinas indicou que com a diminuição da temperatura a dispersão de raios de giro aumenta, indicando a formação de agregados. Para o sistema do Proteassomo, diversas análises se mostraram possível e resultados novos puderam ser obtidos e publicados. Dos estudos utilizando MET,com as micrografias obtidas tem-se a indicação de diferenças na estrutura, quando a molécula está em condições diferentes. Das análises feitas por SAXS, é possível utilizar duas modelagens diferentes, uma simples, outra mais avançada, em que se pode concluir que a redução do Cys-PT glutatiolado induz mudanças conformacionais. Além disso, resultados de SAXS e MET estão em concordância e fornecem informações complementares. / In this work results will be presented on three different protein system: Lysozime, Cryoglobulin and Proteasome, analyzed mainly by Small Angle X-ray scattering technique. Techniques such as transmission electron microscopy and dynamic light scattering were used as complementary techniques. The results show that an unic potential Small Angle X-ray scattering technique around the study of systems in solution. The data correspond to a study of a wide range of sizes for the studied proteins (15kDa to 750 kDa), requiring different approaches in each case. As will be shown for cases where there is a monodisperse system, different modeling methodologies may be used. For the system composed of Lysozyme, because it is a protein widely studied in the literature, it is interesting to compare the results with those already published and observe its stability in solution. The molecular weight calculated from the scattering data was 15kDa, which is in good agreement with the expected for this protein (14.6kDa). Futhermore by varying the concentration of the protein in solution, it is possible to observe a factor of interaction between the particles for higher concentrations. This interaction factor can be considered close to the rigid spheres. For Cryoglobulins system, there was a difficulty in the sample purification, but still present some interesting results, where the SAXS technique provides information on the flexibility of proteins and the analysis for the samples from a pool of immunoglobulin indicated that with decreasing temperature, the dispersion of radius of gyration increases, indicating the formation of aggregates. For the proteasome system, various analyzes have proved possible and new results could be obtained and published. From studies using TEM, with the micrographs we have the indication of differences in the structure, when the molecule is in different conditions. From the analyzes made by SAXS, it is possible to use two different modeling, a simple one, and other more advanced, where it can be concluded that the reduction of the Cys-PT glutathiolated induces conformational changes. Moreover, SAXS and TEM results are in agreement and provide additional information.

The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.

La mitophagie, la dégradation sélective des mitochondries par autophagie, est impliquée dans l’élimination des mitochondries endommagées ou superflues et requiert des régulateurs et protéines spécifiques. Chez la levure, Atg32, localisée dans la membrane externe mitochondriale, interagit avec Atg8, et permet le recrutement des mitochondries et leur séquestration à l’intérieur des autophagosomes. Atg8 est conjuguée à de la phosphatidyléthanolamine et est ainsi ancrée aux membranes du phagophore et des autophagosomes. Chez la levure, plusieurs voies de synthèse de PE existent mais leur contribution dans l’autophagie et la mitophagie est inconnue. Dans le premier chapitre, nous avons étudié la contribution des différentes enzymes de synthèse de PE, dans l’induction de l’autophagie et la mitophagie et nous avons démontré que Psd1, la phosphatidylsérine décarboxylase mitochondriale, est impliquée dans la mitophagie seulement en condition de carence azotée alors que Psd2, localisée dans les membranes vacuolaires, endosomales et de l’appareil de Golgi, est nécessaire en phase stationnaire de croissance. Dans le second chapitre, la relation entre Atg32, la mitophagie et le protéasome a été étudiée. Nous avons démontré que l’activité du promoteur d’ATG32 et la quantité de protéine Atg32 exprimée sont inversement régulées. En phase stationnaire de croissance, l’inhibition du protéasome empêche la diminution de l’expression d’Atg32 et la mitophagie est stimulée. Nos données montrent ainsi que la quantité d’Atg32 est reliée à l’activité du protéasome et que cette protéine pourrait être ubiquitinylée. Dans le troisième chapitre, nous nous sommes intéressés au rôle potentiel de Dep1, un composant du complexe nucléaire Rpd3 d’histones déacétylases, dans la mitophagie. Dans nos conditions, Dep1 semble être mitochondriale et elle est impliquée dans la régulation de la mitophagie. BRMS1L (Breast Cancer Metastasis suppressor 1-like) est l’homologue de Dep1 chez les mammifères. Cette protéine possède un rôle anti-métastatique dans des lignées de cancer du sein. Nous avons trouvé que l’expression de BRMS1L augmente en présence de stimuli pro-mitophagie. / Mitophagy, the selective degradation of mitochondria by autophagy, is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with Atg8, promoting mitochondria recruitment to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to phosphatidylethanolamine (PE). In yeast, several PE synthesis pathways have been characterized, but their contribution to autophagy and mitophagy is unknown. In the first chapter, we investigated the contribution of the different enzymes responsible for PE synthesis in autophagy and mitophagy and we demonstrated that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only in nitrogen starvation, whereas Psd2, located in vacuole/Golgi apparatus/endosome membranes, is required preferentially for mitophagy induction in stationary phase of growth. In the second chapter, we were interested in the relationship between Atg32, mitophagy and the proteasome. We demonstrated that ATG32 promoter activity and protein expression are inversely regulated. During stationary phase of growth, proteasome inhibition abolishes the decrease in Atg32 expression and mitophagy is enhanced. Our data indicate that Atg32 protein is regulated by the proteasome activity and could be ubiquitinated. In the third chapter, we investigated the involvement of Dep1, a member of the nuclear Rpd3L histone deacetylase complex, in mitophagy. In our conditions, Dep1 seems to be located in mitochondria and is a novel effector of mitophagy both in nitrogen starvation and stationary phase of growth. BRMS1L (Breast Cancer Metastasis suppressor 1-like) is the mammalian homolog of Dep1 and has been described in breast cancer metastasis suppression. We found that BRMS1L protein expression increases upon pro-mitophagy stimuli.

Biological processes depend upon the structural and functional quality of the molecules that comprise living organisms. The integrity of molecules such as DNA, RNA, proteins, carbohydrates and lipids is crucial and the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Within a cell, each protein must be present at a specific concentration during certain specific conditions. To maintain cellular homeostasis and the ability to respond to the environment the proteome is in a dynamic state of synthesis and degradation. In eukaryotic cells the ubiquitin-proteasome pathway is the principal mechanism for regulated protein turnover in both the cytoplasm and the nucleus. The 20S proteasome is a cylindrical multi-subunit protease. Proteasomes play an essential role in the targeted and timely ordered degradation of key regulatory proteins and their inhibitors. The 26S proteasome is a 2.500 kDa complex composed of the 20S proteasome sandwiched between two 19S regulators. This is the enzymatic complex responsible for ATP-dependent ubiquitin mediated protein degradation. A polyubiquitin chain attached to a protein serves as a general recognition signal for destruction via the 26S proteasome. It is known that the 19S regulator confers ubiquitin recognition and substrate unfolding to the 20S proteasome, however, the specific functions for many of the different subunits within the 19S complex are not known. We have used RNA interference to study the S13/Rpn11 and S5a/Rpn10 subunits of Drosophila melanogatser proteasomes. We have produced stable cell lines with the human S13 gene under inducible promoters that was used to rescue the knockdown phenotype after RNA interference. The rescue was successful in demonstrating that the human protein is a functional homologue to the Drosophila protein. We call the technique RNAi+c (RNA interference + complementation). This procedure enabled us to also test different mutants of the human S13 protein for their ability to function in the proteasome. Using RNA interference to a Drosophila proteasome subunit in combination with complementation with a corresponding human protein we have been able to study residues important for the deubiquitinating activity of this subunit (Paper I). Interestingly, upon a decrease of either S13 or S5a we see an induction in the levels of active 20S proteasomes. Increase in the levels of the non-targeted 19S subunit can be detected when RNAi treatment is carried out on either S13 or S5a. We have used RNA interference and proteasomal inhibition together with whole genome microarray analysis to reveal a co-regulated network of proteasome genes. This network likely contributes to an overall regulatory system that maintains proper proteasome levels in the cell. Initial studies of the mechanism of transcriptional co-regulation of proteins involved in the 26S proteasome pathway were also performed (Paper II). Finally, the biological function of the proteasome regulator PA28g/REGg is not known. We have studied this regulator in Drosophila using RNA interference and promoter mapping (Paper III).

Protein quality control systems protect cells against the accumulation of toxic misfolded proteins by promoting their selective degradation. Malfunctions of quality control systems are linked to aging and neurodegenerative disease. Folding of polypeptides is facilitated by the association of 70 kDa Heat shock protein (Hsp70) molecular chaperones. If folding cannot be achieved, Hsp70 interacts with ubiquitylation enzymes that promote the proteasomal degradation of the misfolded protein. However, the factors that direct Hsp70 substrates toward the degradation machinery have remained unknown. Here, we identify Fes1, an Hsp70 nucleotide exchange factor of hitherto unclear physiological function, as a cytosolic triaging factor that promotes proteasomal degradation of misfolded proteins. Fes1 selectively interacts with misfolded proteins bound by Hsp70 and triggers their release from the chaperone. In the absence of Fes1, misfolded proteins fail to undergo polyubiquitylation, aggregate, and induce a strong heat shock response. Our findings reveal that Hsp70 direct proteins toward either folding or degradation by using distinct nucleotide exchange factors.

Heat shock proteins (HSPs) are stress-inducible and evolutionarily conserved molecular chaperones that are involved in protein binding and translocation. As molecular chaperones, HSPs bind to denatured proteins, inhibit their aggregation, maintain their solubility, and assist in refolding. This process inhibits the formation of protein aggregates which can be lethal to the cell. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most non-native proteins. Furthermore, proteasome inhibition has been shown to induce hsp gene expression. Celastrol, a quinone methide triterpene, was shown to have an inhibitory effect on proteasome function in mammalian cells. The present study determined that celastrol induced the accumulation of ubiquitinated proteins and reduced proteasomal chymotrypsin-like activity in Xenopus laevis A6 kidney epithelial cells. In addition, incubation of A6 cells with celastrol induced the accumulation of HSP30 and HSP70 in a dose- and time-dependent manner with maximal levels of HSP accumulation occurring after 18 h of exposure. In A6 cells recovering from celastrol, the relative levels of HSP30 and HSP70 accumulation remained elevated for 18-24 h after removal of celastrol. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in celastrol-induced hsp gene expression in A6 cells, since the HSF1 inhibitor, KNK437, repressed the accumulation of HSP30 and HSP70. Exposure of A6 cells to simultaneous celastrol and mild heat shock treatment enhanced the accumulation of HSP30 and HSP70 to a greater extent than the sum of both stressors individually. Additionally, concurrent treatment of A6 cells with low concentrations of both celastrol and MG132 produced different patterns of HSP30 and HSP70 accumulation. While combined treatment with celastrol and MG132 acted synergistically on HSP30 accumulation, relative levels of HSP70 were similar to those observed with MG132 alone. Immunocytochemical analysis of celastrol- or MG132-treated A6 cells revealed HSP30 accumulation in a punctate pattern primarily in the cytoplasm with some staining in the nucleus. Also, in some cells treated with celastrol or MG132 large HSP30 staining structures were observed in the cytoplasm. Lastly, exposure of A6 cells to celastrol induced rounder cell morphology, reduced adherence and disorganization of the actin cytoskeleton. In conclusion, this study has shown that celastrol inhibited proteasome activity in amphibian cultured cells and induced HSF1-mediated expression of hsp genes.

Heat shock proteins (HSPs) are stress-inducible and evolutionarily conserved molecular chaperones that are involved in protein binding and translocation. As molecular chaperones, HSPs bind to denatured proteins, inhibit their aggregation, maintain their solubility, and assist in refolding. This process inhibits the formation of protein aggregates which can be lethal to the cell. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most non-native proteins. Furthermore, proteasome inhibition has been shown to induce hsp gene expression. Celastrol, a quinone methide triterpene, was shown to have an inhibitory effect on proteasome function in mammalian cells. The present study determined that celastrol induced the accumulation of ubiquitinated proteins and reduced proteasomal chymotrypsin-like activity in Xenopus laevis A6 kidney epithelial cells. In addition, incubation of A6 cells with celastrol induced the accumulation of HSP30 and HSP70 in a dose- and time-dependent manner with maximal levels of HSP accumulation occurring after 18 h of exposure. In A6 cells recovering from celastrol, the relative levels of HSP30 and HSP70 accumulation remained elevated for 18-24 h after removal of celastrol. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in celastrol-induced hsp gene expression in A6 cells, since the HSF1 inhibitor, KNK437, repressed the accumulation of HSP30 and HSP70. Exposure of A6 cells to simultaneous celastrol and mild heat shock treatment enhanced the accumulation of HSP30 and HSP70 to a greater extent than the sum of both stressors individually. Additionally, concurrent treatment of A6 cells with low concentrations of both celastrol and MG132 produced different patterns of HSP30 and HSP70 accumulation. While combined treatment with celastrol and MG132 acted synergistically on HSP30 accumulation, relative levels of HSP70 were similar to those observed with MG132 alone. Immunocytochemical analysis of celastrol- or MG132-treated A6 cells revealed HSP30 accumulation in a punctate pattern primarily in the cytoplasm with some staining in the nucleus. Also, in some cells treated with celastrol or MG132 large HSP30 staining structures were observed in the cytoplasm. Lastly, exposure of A6 cells to celastrol induced rounder cell morphology, reduced adherence and disorganization of the actin cytoskeleton. In conclusion, this study has shown that celastrol inhibited proteasome activity in amphibian cultured cells and induced HSF1-mediated expression of hsp genes.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 205-233).