Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

<p>GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. These data suggest that GRP94 is required for important developmental processes relying on cell-cell communication and cell-cell interaction. Consistent with this hypothesis, loss of GRP94 expression in mouse is embryonic lethal yet tissue culture cells expressing no GRP94 are viable. To date, functional studies of GRP94 have relied on cell-autonomous model systems, the use of which has lead to discoveries of proteins that GRP94 chaperones also called client proteins. These systems give limited insight into the essential role(s) played by GRP94 in metazoan biology. The dichotomy that GRP94 is necessary for metazoan life, but dispensable for cellular viability suggests that the chaperone is required for the functional expression of secretory and/or membrane proteins that enable cells to function in the context of tissues.</p><p>To explore this hypothesis, the <italic>Drosophila</italic> ortholog of <italic>GRP94</italic>, <italic>Gp93</italic>, was identified and <italic>Gp93</italic> mutants were created using imprecise P-element excision. <italic>Gp93</italic> was found to be an essential gene in <italic>Drosophila</italic>. Loss of zygotic <italic>Gp93</italic> expression is late larval lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. <italic>Gp93</italic> mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of <italic>Gp93</italic>-expression are profound; <italic>Gp93</italic> mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of <italic>Gp93</italic> expression resemble those reported for mutations in <italic>labial</italic>, an endodermal homeotic gene required for copper cell specification, and &alpha;-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding labial protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton.</p><p>The creation of <italic>Gp93</italic> mutant <italic>Drosophila</italic> has allowed for the study of GRP94 function <italic>in vivo</italic> and will be of upmost importance to future studies examining the function of this chaperone in all aspects of metazoan biology. This dissertation focuses on the morphological and physiological defects that accompany loss of <italic>Gp93</italic> expression in <italic>Drosophila</italic> larvae. It will also outline future studies utilizing this model.</p> / Dissertation

Glaucoma is a neurodegenerative protein misfolding disorder classified by increases in IOP, damage to retinal ganglion cells (RGCs), optic nerve (ON) head damage, and progressive irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma, constituting over 90% of clinical cases. POAG is observed in patients where normal outflow channels, mainly the trabecular meshwork (TM), are exposed at the angle formed by the iris and cornea. However, due to TM cellular dysfunction, aqueous outflow resistance is increased preventing normal circulation of aqueous humor. Recent studies have shown that in 2-4% of POAG cases, increased intracellular levels of a secreted glycoprotein, called myocilin, are present in the TM. Myocilin is a 504aa glycoprotein, with an unknown precise function. Recent studies have postulated the importance of myocilin in oligodendrocyte differentiation, axonal myelination, and early apoptosis of retinal cells in development, but exact function is still widely debated. However, it is important to note that pathology associated with myocilin is only seen during POAG. Also, only cells of the TM exhibit toxicity when overexpressing mutant myocilin. In the normal eye, myocilin is secreted from the ER of TM cells, however, mutations in the MYOC gene lead to an aggregation-prone form of the protein, which is inefficiently processed and degraded from the ER, leading to build-up and associated toxicity. There are over 70 known MYOC mutations associated with glaucoma, with over 90% occurring in the C-terminal OLF domain. Some of the more common, pathological mutations are: I477N, Y437H, P370L, W286R, N480K. All of these mutations have been observed in patients with glaucoma, and all lead to build-up and aggregation of the mutant protein within the ER of TM cells. Recently, work out of our lab has discovered an interaction between mutant forms of myocilin and the chaperone Grp94. Grp94 is the resident Hsp90 isoform of the ER. Grp94 is an important chaperone in ER quality control, aiding in the output of properly folded secretory and membrane-bound proteins. Besides protein folding, other roles of Grp94 in the ER include: calcium buffering, roles in ER quality control (including targeting misfolded proteins for ERAD), peptide binding, and roles in ER stress. Generally, terminally misfolded proteins in the ER are degraded through ERAD; the Grp94 mediated shuttling of misfolded proteins to the ER trans-membrane machinery for ubiquitination and subsequent translocation to the cytosol for proteasomal degradation. However, in the case of POAG, ERAD is inefficient in mutant myocilin degradation, causing protein accumulation within the ER. In this study, we demonstrate that specific Grp94 inhibition of interaction with mutant or misfolded myocilin leads to myocilin degradation and subsequent lowering of protein accumulation in the TM, thus reducing downfield pathology associated with POAG. Grp94 preserves mutant myocilin in the ER of TM cells leading to protein accumulation and aggregation precipitating TM cellular dysfunction. We showed in various in vitro cell assays that Grp94 inhibition leads to a reduction in intracellular protein levels, while alleviating TM cellular toxicity. Furthermore, in the Tg-MYOCY437H mouse model of POAG, we showed that topical ocular administration of a specific Grp94 inhibitor alleviated glaucomatous pathologies, including elevated IOP, myocilin accumulation in the TM, reduced scotopic/photopic visual responses, and RGC health and viability. Finally, we have proven the importance of ER-stress pathway malfunction in the development of POAG pathology, while also discovering the involvement of the autophagy mechanism for myocilin degradation following Grp94 inhibition. Overall, this work proves that Grp94 is an important regulator of myocilin pathology during POAG. While there are no current therapeutics on the market that directly target the underlying POAG disease mechanism, specific Grp94 inhibition shows great promise and should be considered for human clinical trials. If successful, specific targeted Grp94 inhibition could be the first curative therapeutic options for patients suffering from myocilin-associated POAG.

No description available.

Heat shock proteins (HSPs) are expressed in response to environmental stresses. Compared to other kingdoms, plant HSP families are larger, presumably the result of adaptation to a wide range of stresses. Following on an analysis of drought stress characteristics in loblolly pine (Watkinson et al., 2003), expression patterns of HSP gene expression during photosynthetic acclimation were examined. One cycle of mild (-1Mpa) followed by two cycles of severe stress (-1.7Mpa) were probed for conditioning effects. Photosynthetic acclimation occurred after the first cycle. No acclimation occurred without the first mild cycle. Microarray/RT-PCR analyses showed that a pine homolog to GRP94 (ER-resident HSP90) was up-regulated after rehydration coincident with acclimation. This GRP94 is closely related to GRP94 from the desiccation tolerant plant X. viscosa, supporting the importance of this gene during acclimation to water deficit. HSP genes whose products localized to the mitochondrion showed gradual up-regulation after consecutive cycles of severe drought. The Arabidopsis pine GRP94 homolog, (AtHSP90-7) was then analyzed, using bioinformatics (Pati et al., 2006) and laboratory tools. Genes encoding putative candidate co-chaperones for GRP94 and other HSP90s were discovered, which contained water stress-related cis-elements. Arabidopsis (Col-0) wild type and two T-DNA insertion mutants in HSP90-7 were used to study the importance of this gene for photosynthetic acclimation. Only the mutants were able to acclimate to drought stress, with the level of AtHSP90-7 expression in the mutants being reduced compared to the wild type. AtHSP90-7 may have a different role in Arabidopsis, and its reduced expression activated other protective genes (Klein et al., 2006). Responses to extreme drought in resistant (Sullu) and susceptible (Negra Ojosa) lines of Andean potatoes were also compared in order to identify relationships between HSPs gene expression, and tolerance, defined as the ability to maintain photosynthesis at 50% after 25 days of drought and to recover from the stress. Tolerance was correlated with up-regulation of HSPs (mostly chaperonins) and antioxidant genes all of whose gene products are located in the chloroplast. / Ph. D.

No description available.

<p>Extracellular GRP94 can elicit both innate and adaptive immune responses by interacting with endocytic and signaling receptors on professional antigen presenting cells (pAPCs). CD91 was the first receptor proposed to facilitate GRP94-mediated immune responses. Using a GRP94 affinity matrix, a CD91 fragment was isolated from the detergent-solubilized membranes of a pAPC cell line. It was then demonstrated that CD91 ligands could inhibit GRP94-mediated peptide cross-presentation, suggesting that CD91 played a critical role in this process. While these studies implied that CD91 could function as a GRP94 endocytic receptor, later works suggested that CD91 may not recognize GRP94 at the cell surface. These opposing observations have lead to a significant controversy surrounding the identity of CD91 as an endocytic receptor for GRP94. Because the ability of CD91 to directly mediate GRP94 surface binding and uptake has not been established, the studies included in this dissertation have focused on evaluating the ability of CD91 to facilitate three processes that are necessary for GRP94-mediated peptide cross-presentation: surface binding, internalization, and processing.</p><p>These studies utilized a recombinantly-expressed N-terminal domain of GRP94 (GRP94.NTD), which was previously shown to have nearly identical biological activity to full length GRP94. The ability of CD91 to directly bind and internalize GRP94.NTD was examined using murine embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via siRNA, or eliminated by genetic disruption of the CD91 locus. Binding competition experiments were also conducted. Together, these studies reveal that CD91 does not directly interact with GRP94 at the cell surface. The ability of CD91 to directly facilitate GRP94 internalization was examined using various internalization and internalization competition assays. These studies demonstrated that GRP94.NTD and the CD91 ligand RAP were internalized through spatially and kinetically distinct pathways, that CD91 was not necessary for GRP94.NTD internalization, and that RAP did not inhibit GRP94 endocytosis. Together, these studies strongly suggest that CD91 does not directly facilitate GRP94 internalization. When these studies were extended to DC2.4 mouse dendritic cells, the CD91 ligand RAP reduced GRP94.NTD internalization/process by ~15%. This suggests that CD91 may indirectly facilitate GRP94 internalization in pAPC cell lines. Lastly, cross-presentation studies were utilized to examine the ability of various CD91 ligands to influence GRP94.NTD-mediated peptide cross-presentation through a post-uptake mechanism using the DC2.4/OT-1 system. Although it was discovered that DC2.4 cells can internalize and process GRP94.NTD/peptide complexes through fluid-phase endocytosis, CD91 ligands did not significantly inhibit GRP94-mediated peptide cross-presentation by DC2.4 cells. These studies demonstrate that CD91 does not play a primary role in GRP94-mediated peptide cross-presentation.</p><p>In the course of these studies, cell surface heparan sulfate proteoglycans (HSPGs) were identified as novel cell surface binding sites for GRP94.NTD on MEF cells. This conclusion was established using three distinct experimental approaches. GRP94.NTD surface binding was significantly decreased following heparin pre-treatment, following incubation with the sulfation inhibitor sodium chlorate, and following digestion with extracellular heparinase II. Conversely, these treatments did not significantly influence GRP94.NTD binding to RAW264.7 mouse macrophage-like cells. This suggested that GRP94.NTD-HSPG cell surface interactions may require the expression of a specific type of cell surface HSPG that is not expressed by RAW264.7 cells. However, additional studies strongly suggested that GRP94.NTD-HSPG cell surface interactions were mediated by the heparan sulfate-containing side chains rather than the presence of a specific cell surface HSPG core protein.</p><p>This dissertation focuses on the critical re-examination of CD91 functions in GRP94 surface binding, uptake, and cross-presentation. Together, these results clarify conflicting data on CD91 function in GRP94 surface binding and endocytosis. This dissertation also describes the identification of cell surface HSPGs as GRP94 binding sites on MEF cells. These studies extend the diversity of surface receptors that recognize of GRP94, and suggest that cell surface HSPG-dependent interactions may contribute to the biology of GRP94-elicited immune responses.</p> / Dissertation