Polyglutamine-expanded fragments, derived from the human huntingtin protein, are aggregation-prone and toxic in yeast cells, bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline-rich region targets polyglutamine aggregates to the large perinuclear deposit (aggresome). Aggresome targeting ameliorates polyglutamine cytotoxicity in the presence of the prion form of Rnq1 protein, however, aggresome-forming construct remains toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). Disomy by chromosome II partly ameliorates polyglutamine toxicity in the strains containing Sup35 prion. The chromosome II gene, coding for another release factor, and interaction partner of Sup35, named Sup45 (eRF1), is responsible for amelioration of toxicity. Plasmid-mediated overproduction of Sup45, or expression of the Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into prion aggregates, also ameliorate polyglutamine toxicity. Protein analysis indicates that polyglutamines alter aggregation patterns of the Sup35 prion and promote aggregation of Sup45, while excess Sup45 counteracts these effects. In the absence of Sup35 prion, disomy by chromosome II is still able to decrease polyglutamine toxicity. However, SUP45 is no longer the gene responsible for such an effect. Taken together with the finding that the presence of both the Rnq1 prion and the Sup35 prion has an additive effect on polyQ toxicity, one gene or few genes on chromosome II are able to ameliorate polyQ toxicity through a SUP45-independent pathway. The identification of such a gene is currently ongoing. Monosomy by chromosome VIII in diploid heterozygous by AQT (Anti-polyQ Toxicity mutants that are disomic by chromosome II) counteracted the effect of AQT. Similarly, deletion of the arg4 gene in chromosome VIII in AQT haploid was able to eliminate the AQT effect. Moreover, analysis of genes involved in the arginine and polyamine synthesis indicated that loss of genes in later stages of arginine biosynthesis causes increase of polyglutamine toxicity. Deletion of genes arg1, arg4, arg8 (arginine pathway) and spe1 (polyamine pathway) all suppressed the Sup35 prion phenotype expression in the nonsense suppression system. Further analysis regarding the mechanisms behind those effects is needed. Our data uncover the mechanisms by which genetic and epigenetic factors may influence polyglutamine toxicity, and demonstrate that one and the same type of polyglutamine deposits could be cytoprotective or cytotoxic, depending on the prion composition of a eukaryotic cell.

This dissertation is focused on developing cross-linking and mass spectrometry methodologies to study protein-protein interactions. Top-down cross-linking, in combination with mass spectrometry, provides advantages over bottom-up approaches, such as retaining posttranslational modification. Intermolecular cross-linking studies focus on defining protein complex topology and protein-protein interactions. We first developed the top-down MS approach to analyze intermolecular cross-linking in human hemoglobin. Both α-α and β-β intermolecular cross-linking were found and the cross-linking sites on the protein were identified, obtaining distance constraints between subunits of the human hemoglobin protein complex. This methodology would be a promising approach to characterize protein complexes and protein-protein interactions with high throughput and automation. This dissertation also focuses on development of cross-linking mass spectrometry to study synphilin-1 interactors and aggresome formation. Synphilin-1 is a protein that promotes the formation of protein aggregates and aggresome formation upon proteasome inhibition, and is implicated in Parkinson disease. Synphilin-1 contains several protein binding motifs. The biological functions of synphilin-1 and its role in aggresome formation and the pathogenesis of Parkinson disease remain to be elucidated. We utilized tandem affinity purification and label-free mass spectrometry to explore the patterns of cellular proteins associated with synphilin-1. We identified 57 synphilin-1 interacting proteins, and functional enrichment and pathway analysis showed that many of the associated proteins are involved in chromatin modulation, RNA and protein metabolism. Furthermore, we developed a proteomic strategy to identify synphilin-1 binary interacting partners as well as interacting domains using affinity purification followed by isotopically tagged cross-linking in combination with mass spectrometry. We found 24 newly discovered proteins that directly bind to synphilin-1. The proteins were mainly involved in RNA metabolism. The coiled-coil domain (CC), ankyrin-like repeat domain 2 (ANK2), and the protein aggregate promoting domain, appeared to the main regions that bound proteins. The functions of synphilin-1 interacting proteins, such as CK2, in aggresome formation were studied. The results show that CK2 is an important regulator of aggresome formation, but not through its kinase activities. Involvement of synphilin-1 in autophagy was also investigated. Knockdown of synphilin-1 shows that synphilin-1 impacts autophagy.

L'étiologie de la forme sporadique de la maladie d'Alzheimer (AD) reste largement inconnue. Toutefois, une adéquation entre des facteurs environnementaux et génétiques est fortement probable. C'est ainsi que de nombreux arguments suggèrent que le virus herpes simplex de type 1 (HSV1) en infectant et en se répliquant dans le système nerveux central, puisse être un co-facteur impliqué dans le déclenchement et le développement de l'AD. Pour éprouver cette hypothèse, nous avons développé un modèle cellulaire constitué de neurones de rat infectés par HSV1 pour analyser les modifications viro-induites de leur expression génique. Il a été mis en évidence dans les neurones infectés : i) une augmentation de la production du peptide amyloïde Aβ42 et de Tau phosphorylée, ainsi que leur agrégation dans un agrésome intracellulaire ; ii) des variations du niveau de transcription de nombreux gènes très similaires à celles observées chez des patients AD. Par ailleurs, l'étude des mécanismes moléculaires de l'apoptose viro-induite dans ce modèle original a permis de mettre en évidence une corrélation entre l'activation des caspases et la production d'Aβ42 et une corrélation entre le phénomène d'apoptose avortée (abortosis) et la formation d'agrésome. De l'ensemble des ces résultats, il apparait que ce modèle cellulaire est représentatif de certains aspects des stades précoces de l'AD et conforte l'hypothèse qu'HSV1 serait un co-facteur dans la maladie d'Alzheimer / The origin of the sporadic form of the Alzheimer's disease (AD) remains still widely unknown. However, an adequacy between environmental and genetic factors is highly probable. Numerous arguments suggest that the virus herpes simplex of type 1 (HSV1) by infecting and replicating in the central nervous system, could be a co-factor involved in the AD process. To evaluate this hypothesis, we set up a model made of rat neurons infected by HSV1 in order to analyse the virally-induced modifications of their gene expression. Using this model we have shown: i) an over-production of the amyloid peptide Aß42 and of phosphorylated form of Tau accompanied by their concentration within an intracellular aggresome; ii) variations of the transcription levels of numerous genes equivalent to that observed in AD patients. Furthermore, the study of the molecular mechanisms underlying the virally-induced apoptosis allowed to point out a correlation between caspase activation and Aß42 production as well as a correlation between abortosis and aggresome formation. All together these results demonstrate that this cellular model represents, at least in part, some aspects of the early stages of AD and bring evidences that HSV1 could be a co-factor in the AD process

Indiana University-Purdue University Indianapolis (IUPUI) / Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.