Not available / text

The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in all eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via three GTPases; Gtr1, Gtr2, and Rho1, and the SEA-associated Npr2/3 proteins. However, little is known about the way that other nutrient or stress signals are transmitted to TORC1. Here I present two studies identifying how, and at what level, glucose and other environmental stimuli act to tune TORC1 signaling. In the first study I show that the TORC1 pathway populates three additional stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. The TORC1 pathway remains in the glucose starvation state even when cells are simultaneously starved for nitrogen and glucose or treated with rapamycin. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, the data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell. In the second study I investigate further the observed hierarchy of TORC1 inputs. I show that glucose starvation triggers disassembly of TORC1, and movement of the key TORC1 component Kog1, to a single body near the edge of the vacuole. These events are driven by AMPK/Snf1-dependent phosphorylation of Kog1 at Serine 491/494 and two nearby prion-like motifs. Kog1-bodies then serve to increase the threshold for TORC1 activation in cells that have been starved for a significant period of time. Together, this data shows that Kog1-bodies create hysteresis (memory) in the TORC1 pathway and help ensure that cells remain committed to a quiescent state under suboptimal conditions.

To thrive when conditions are favorable and survive when they are stressful, cells must carefully regulate their growth rate and stress response programs. This requires rapid, coordinated regulation of many genes in response to information about the levels of numerous nutrients and stress conditions. We are beginning to understand how, in eukaryotes, the TORC1 and PKA pathways regulate growth in nutrient rich conditions. However, how cells tune growth and stress responses in suboptimal conditions is largely unknown. To address this, we ran screens to begin reconstructing the growth regulation network in stress conditions. We found many novel regulators, including signaling proteins, components of the vacuolar ATPase, transcription factors, and components of the endomembrane system. In order to place these regulators in the TORC1 pathway, we performed follow up experiments on over 300 of these regulators using the TORC1 inhibitor rapamycin. We were able to place many new components in the TORC1 pathway, including 59 genes that act downstream of TORC1. We were particularly interested in the discovery that Vip1, a conserved inositol pyrophosphate kinase, was necessary for the shutdown of hundreds of growth genes in stress and starvation conditions. In subsequent experiments, we learned that the inositol pyrophosphate second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are critical regulators of cell growth and the general stress response, acting in parallel to the TORC1 pathway to control the activity of the class I HDAC Rpd3L. Taken together, this work reveals many new regulators of cell growth and shows how delineation of one such regulator uncovered a global role for a little known family of second messengers.

Thioredoxin-1 is a redox protein upregulated in many cancers. Its functions include inhibition of apoptosis, increasing cellular growth and proliferation. It has been shown that cells displaying increased levels of Trx-1 have increased drug resistance. PX-12 is a Trx-1 inhibitor that shows anti-proloferative and cytotoxic activity in vitro and in vivo. We used surface enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) to measure plasma Trx-1 levels of patients treated with PX-12 as a side study of a phase-I trial. SELDI-TOF-MS was able to measure a decrease in plasma Trx-1 after PX-12 treatment semi-quantitatively. In addition, SELDI measured 57 other protein peaks in plasma; seven which were found in all plasma samples analyzed. One of these peaks was located at 13.86kDa and identified through LC-MS/MS sequencing to be a variant of Transthyretin. Further studies into these additional peaks are necessary to determine their biological importance in relation to Trx-1 and PX-12.

<p> Endothelial cells are a vital region in the pathophysiology of the vasculature because it is the interface between blood flow and the vessel. One way that the structure of the vessels wall can change is by the accumulation of reactive oxygen species (ROS), which has been correlated to aneurysm formation. Four main ROS sources in endothelial cells are: NADPH oxidase, mitochondria electron transport chain, eNOS uncoupling, and xanthine oxidase. Endothelial cells are an essential component of vasculature that has distinct functions and morphology. The aorta and brain arteries are highly populated by endothelial cells but the morphology and cellular signaling has been shown to be different. This study focuses on the difference between brain and aorta ROS production and how flow affects ROS. Joeseph Moran-Guiati and Jason Kushner provided the brain and aortic endothelial cultures for these studies. NADPH oxidase complex is the main contributor in both cell types but more in brain. Surprisingly, both cell types contain approximately the same number of NOX subunits, suggesting that the difference in ROS production is dependent on how activated these subunits are. Mitochondrial ROS was only significantly generated in brain cells and is verified because brain endothelium contains higher numbers of mitochondria. Both uncoupling of eNOS and xanthine oxidase did not contribute to ROS generation in static cultures. ROS production increased even further in both cell types when cells were exposed to flow and even higher in brain, suggesting that flow effects ROS generation. These results provide useful information in the difference between ROS generation and how it can be harmful in possibly causing intracranial aneurysm formation.</p>

Proliferating cells have unique metabolic requirements beyond those of quiescent cells. Specifically, blood forming hematopoietic stem cells, during periods of severe blood loss, switch from a quiescent glycolytic state to a state dependent on mitochondrial metabolism during differentiation and proliferation. This dissertation attempts to define some of the signaling details of this switch by using erythropoietin receptor signaling as a model. In cytokine-dependent Ba/F3 cell line expressing the receptor for erythropoietin (EpoR) (Ba/F3-EpoR), chemical inhibition of mitochondrial function by rotenone decreases in erythropoietin(Epo)-stimulated proliferation. This observation led to the examination of whether Epo could stimulate mitochondrial function. To further assess the role of mitochondria in cell proliferation and the metabolic functions of Epo, levels of oxidative phosphorylation markers and signaling molecules important for mitochondrial biogenesis were measured. Western blotting scans showed increased protein levels of cytochrome oxidase subunit IV (CoxIV) and Complex III core protein 2 following 24 hours of Epo treatment. Interestingly, inhibition of Janus Kinase 2 (Jak2), the tyrosine kinase associated with Epo receptor, by AG490 elicited a similar decrease in CoxIV to Epo withdrawal even in the presence of Epo. In addition, Epo increased the levels of the mitochondrial biogenesis regulator AMP-activated protein kinase α (AMPKα) in a Jak2-dependent manner within Ba/F3 cells. Both total and phosphorylated (activated) AMPKα were increased following Epo stimulation. Treatment with the AMPK inhibitor Compound C decreased Epo stimulation of CoxIV, suggesting a linear signaling cascade from Jak2 to mitochondrial biogenesis through AMPKα. Examining potential mechanisms, direct binding of AMPKα to (EpoR) and Jak2 were observed through immunoprecipitations of transfected lysates in a manner exclusive to AMPK regulator subunits β and γ. Furthermore AMPKα was found to be tyrosine phosphorylated in an Epo and Jak2 dependent manner. Taken together, data in this dissertation suggests a role for Epo in regulating mitochondrial biogenesis in cytokine dependent cells through a potential mechanism of forming a signaling complex between EpoR, Jak2, and AMPKα. This signaling complex may provide intersection between Epo's signaling in cell proliferation and metabolism through AMPKα.

Skeletal muscle is composed of repeating units called sarcomeres which contain distinct sets of thin and thick filaments that slide past each other during contraction. In addition to these proteins a third filament called titin acts as a molecular spring and prevents overstretching of muscle. In skeletal muscle, titin's spring-like elements include the PEVK sequence which elongates upon stretch and immunoglobulin-like (Ig) domains. A fourth myofilament, nebulin, is anchored into the Z-disk and present along the length of the thin filament. Nebulin is proposed to be a regulator of thin filament length. These large sarcomeric proteins can be differentially spliced to yield proteins of various sizes and properties. During my graduate career I sought to elucidate the role of titin and nebulin in skeletal muscle development and disease. Firstly, I studied if differential splicing of titin and nebulin occurred during development. Early post-natal development is a time of rapid isoform switching and growth, and I hypothesized that these large proteins could be affected. In post-natal development of the mouse, large compliant titin molecules are gradually replaced with shorter, stiffer isoforms through removal of PEVK exons. In nebulin, C-terminal exons present in the Z-disk are differentially expressed between muscle types and throughout development which correlates with differences in Z-disk width. My research has shown that titin and nebulin transcripts are tuned during development with changes in titin affecting the I-band region of the molecule and changes in nebulin affecting the Z-disk region. Secondly, I sought to study the effect of specific titin domains on titin elasticity in skeletal muscle. Changes in titin's stiffness occur in various myopathies but whether these are a cause or an effect of the disease is unknown. To test this, a genetically engineered mouse model was created in which part of the constitutively expressed immunoglobulin-like (Ig) domains of titin (Ig3-11) were removed. Unexpectedly, the deletion of these domains causes additional differential splicing to take place in skeletal muscle and leads to skeletal muscle myopathy. I sought to investigate the mechanism by which this occurs and found that RBM20, a titin splice factor, was significantly increased in IG KO mice and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Through the use of this model the mechanisms that underlie titin alternative splicing were explored and demonstrated how alternative splicing alters muscle function. My third project was to better understand the mechanisms by which nebulin loss causes the disease nemaline myopathy (NM). We generated a mouse model in which nebulin's exon 55 is deleted (NEBΔex55) to replicate a founder mutation seen frequently in NM patients with Ashkenazi Jewish heritage. The mice phenocopy pathology of severe myopathy with a short lifespan, changes in thin filament length and cross bridge cycling kinetics, and changes in calcium sensitivity. Force generation in this model is improved by the addition of a calcium sensitizer and supports the use of these compounds in treating patients with nemaline myopathy. In my last project, a second mouse model in which nebulin levels are reduced (NEB cKO) was created to study the effects of reduced nebulin levels on survival and pathology of skeletal muscle. NEB cKO mice recapitulate many of the hallmark features of typical congenital NM, and mice have muscle type dependent effects on contractility and trophicity. Most notably, the intact EDL muscle had a 84% reduction in maximal active force compared to that of the soleus muscle which had a 42% reduction in force. These differences can be explained in part by changes in thin filament length, cross bridge cycling kinetics, and muscle fiber disarray. In conclusion, through the use of genetically engineered mouse models, differential splicing of titin and nebulin during development has been characterized and mechanisms by which mutations in these large sarcomeric proteins cause skeletal muscle disease have been elucidated.

Colon cancer is the second leading cause of cancer deaths in the United States of America. It is caused by the accumulation of mutations in tumor suppressors and oncogenes. The APC tumor suppressor is mutated in most diagnosed cases of colorectal cancer. Mutations in the K-RAS oncogene occur at later stages of colon cancer progression. In the present study, the transcriptional regulation of a novel target of these two genes, caveolin-1, was studied. Caveolin-1 is transcriptionally regulated by the APC tumor suppressor gene, via induction of its inducer, FOXO1 and the suppression of its transcriptional repressor, C-MYC. An activated K-RAS oncogene induces caveolin-1 transcription via activation of the P-I3 Kinase pathway. In addition to transcriptional regulation of caveolin-1, the influence of caveolin-1 expression on cellular phenotypes like signal transduction and polyamine uptake were assessed. The present studies demonstrate that caveolin-1 expression affects basal levels of AKT and ERK signaling, with an increased signaling associated with caveolin-1 expression in these colon tumor-derived cells. In addition, caveolin-1 expression positively affects signaling in response to an inflammatory stimulus like TPA. Interestingly, caveolin-1 expression leads to a decrease in the uptake of pro-tumorigenic molecules like polyamines, in the colon cell lines tested. Taken together, the data from this study suggests that caveolin-1 is transcriptionally regulated by the APC and the K-RAS gene at different stages of colorectal tumorigenesis and this in turn, leads to different phenotypes influenced by caveolin-1 expression.

The coordination of various cellular differentiation and morphogenetic programs during plant embryogenesis is required to establish the basic adult body plan. The molecular basis of these patterning events remains to be fully understood. In particular, little is known about the roles of cell-cell signaling during embryonic pattern formation.I identified two receptor-like kinases, RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), redundantly required for Arabidopsis thaliana embryonic pattern formation. Genetic analysis indicates that RPK1 and TOAD2 have overlapping embryonic functions. The zygotic gene dosage of TOAD2 in an rpk1 background is of critical importance, suggesting that signaling mediated by RPK1 and TOAD2 must be above a threshold level for proper embryo development. The localization of RPK1 and TOAD2 translational fusions to GFP coupled with the analysis of cell-type specific markers indicate that RPK1 and TOAD2 are redundantly required for both pattern formation along the radial axis and differentiation of the basal pole during early embryogenesis.I found that RPK1 and TOAD2 also have overlapping functions required for cotyledon primordia initiation during Arabidopsis embryogenesis. Genetic analyses indicate that cotyledon initiation is sensitive to TOAD2 gene dosage in an rpk1 background. Analysis of cell-specific markers suggest that RPK1 and TOAD2 are primarily required for the differentiation of cell types (i.e. the central domain protoderm) subjacent to the cotyledon primordia, and that the cotyledon initiation defects are caused by defects in the central domain protoderm. In addition, RPK1-GFP and TOAD2-GFP translational fusions had overlapping localization patterns in the central domain protodermal cells when cotyledon primordia were first recognizable. I propose that RPK1 and TOAD2 are primarily required to maintain central domain protoderm cell fate and that the loss of this key embryonic cell type in mutant embryos results in patterning defects throughout the embryo including the failure to initiate cotyledon primordia.This work has identified two putative receptors for cell-cell signals that mediate key patterning events during plant embryogenesis. The future identification of components in the RPK1 and/or TOAD2 signaling pathways will yield further insight into the molecular basis of the generation and assembly of diverse embryonic cell types.

Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient <italic>Saccharomyces cerevisiae</italic> deletion strains for defects in mitochondrial RNA processing revealed that two novel genes affect processing of mitochondrial tRNAs by RNase P. One gene encodes Htd2, an enzyme in the type II mitochondrial fatty acid synthesis pathway (FAS II). The other gene is described here as encoding Lip3, an enzyme involved in the synthesis and attachment of the co-factor lipoic acid, which is synthesized from a product of the FAS II pathway.RPM1 is the mitochondrial-encoded RNA subunit of mitochondrial RNase P. The multigenic transcription unit containing RPM1 also contains tRNA<super>pro</super>. Maturation of RPM1 necessitates processing of the tRNA by RNase P. Thus, RNase P is required for maturation of its own RNA component, constituting a positive feedback cycle. The present work demonstrates that a product of the FAS II pathway is necessary for the assembly or activity of RNase P, as deletion of any gene encoding an FAS II enzyme results in inefficient processing of tRNApro from the transcript.Analysis of the enzymes involved in the synthesis and attachment of lipoic acid to target proteins is also described here. Disruption of any of these enzymes affects protein lipoylation and tRNA processing. Gcv3, a target of lipoylation, was found to be required for lipoylation as well as for efficient tRNA processing.A second feedback cycle controlling pyruvate dehydrogenase activity and fatty acid synthesis may be functional under certain conditions. Pyruvate dehydrogenase, which provides acetyl-CoA for the FAS II pathway, requires lipoic acid for its activity. It is hypothesized that the two feedback cycles and the role of Gcv3 may provide switch-like regulation of mitochondrial gene expression in response to the nutritional state of the cell.