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Browsing by Subject "Autophagy"

Now showing 1 - 8 of 8
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    Distinct functions of autophagy kinases ULK1 and ULK2 in adipogenesis and adipocyte metabolism
    (2011-11) Ro, Seung-Hyun
    Autophagy, the catabolic process through which intracellular constituents are degraded in the lysosome under nutrient starvation or stress, has gained growing attention in the field of diabetes and obesity (Goldman S 2010; Ost A 2010; Beau I 2011; Kovsan J 2011). Despite the fundamental cellular function of autophagy in maintaining cellular energy homeostasis and survival under nutrient– or energy– deprived conditions and stress, the role of adipose autophagy in metabolism and metabolic diseases remains largely unknown. The goal of my study has been to better understand the function of autophagy in adipogenesis and in the regulation of adipocyte metabolism. My study has been focused on defining the role of ULK1 (Unc–51 like kinase 1, mammalian homolog of Atg1, hATG1) and its homologue ULK2 in the regulation of adipogenesis, metabolism and mitochondrial functions in adipocytes. ULK1 and ULK2 are key regulators of autophagy induction in mammalian cells (Kundu M 2009; Chang YY 2009; Ganley IG 2009; Hosokawa N 2009; Jung CH 2009). Knockdown of ULK1 or ULK2 inhibited autophagy in 3T3–L1 adipocytes, suggesting that they play important roles in autophagy in adipocytes. The knockdown experiment also revealed that ULK1 and ULK2 share key functions in lipolysis, mitochondrial respiration and protection of cells against oxidative stress. Despite these shared functions, their knockdown had different or even opposing effects on several metabolic parameters. Knockdown of ULK1 raised PPAR–γ level, facilitated differentiation of 3T3–L1 cells, increased the levels of GLUT4, insulin receptorβ(IRβ) and insulin receptor substrate–1 (IRS–1), and insulin–stimulated glucose uptake, and reduced fatty acid oxidation. By contrast, knockdown of ULK2 had opposite or no significant effects on these parameters. Through knocking down both ULK1 and ULK2, we found that ULK2 has a dominant effect over ULK1 in the regulation of adipogenesis. These results demonstrate that ULK1 and ULK2 have distinct functions in the regulation of adipogenesis and adipocyte metabolism, and that ULK2–dependent autophagy appears to be important for adipogenesis.
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    Exploring The Interactions And Functions Of The ULK1 Complex In The Autophagy Pathway
    (2014-02) Otto, Neil
    Autophagy, an evolutionarily conserved process through which cellular components or organelles are degraded through lysosomes, is induced when eukaryotic cells are under nutrient starvation or cellular stress conditions. The ULK1 (UNC-51 like kinase 1) complex consisting of ULK1, Atg13, FIP200, and Atg101 plays a key role in mediating cellular nutritional status to the regulation of autophagy. Despite the recent advance in our understanding of the ULK1 functions, how the ULK1 complex regulates autophagy induction remains unclear. Here, we identify that the ULK1 complex interacts with mammalian Atg8 homologs via Atg13 and the interaction is important for autophagosome formation. Through a yeast two-hybrid screen, we identified a clone harboring the full length GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) as an Atg13 binding protein. Through co-immunoprecipitation and in vitro binding assays, we confirmed that Atg13 directly interacts with GATE-16, as well as GABARAP (Gamma-aminobutyric acid receptor-associated protein) and GABARAPL1 (GABA-A receptor-associated protein-like 1), but not LC3B (Microtubule-associated protein1B-light chain 3), via a conserved LC3 interacting region (LIR) near its C-terminus. The Atg13-Atg8 interaction was greatly increased when cells were induced to accumulate protein aggregates or mitochondrial damage, but not by nutrient starvation, implying that the interaction might respond to selective autophagy inducing conditions. The LIR-disrupting mutation of Atg13 suppressed the degradation of p62/sequestosome 1, poly-ubiquitinated protein aggregates, and damaged mitochondria. These results suggest that Atg13 might participate in autophagy, especially selective autophagy, via interacting with GABARAP subfamily Atg8 proteins. p62 is a protein involved in selective autophagy that also interacts with Atg8 proteins via its LIR motif. My study revealed that ULK1 binds and phosphorylates p62. Several phosphorylation sites of p62 were identified by mass spectrometry. Mutational approaches revealed that some of the identified phosphorylations are important for colocalization of p62 with LC3 and for autophagic clearance of mutant huntingtin aggregates. The culmination of this work suggests that the ULK1 complex recruits GABARAP subfamily proteins and phosphorylates p62 in the pathway of autophagy induction.
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    Interactions between Human Two-pore Channels and Nonaspanin Proteins
    (2016-08) SUN, LEISHENG
    Autophagy is an evolutionary conserved lysosomal degradation pathway which is involved with a variety of cellular process, but the relevant mechanisms remain elusive. Here I find that TM9SF1, a nine-spanning transmembrane protein and human two-pore channels (TPCs) regulate cell autophagy. More importantly, TM9SF1 and TPCs are found in endolysosomal system, and among all TM9SF family, only TM9SF1 interacts with TPC2 structurally, which suggests that there might be functional interaction between TM9SF1 and TPC2. Indeed, my results showed that TM9SF1 increases LC3-II/LC3-I ratio in TPCs knockdown groups significantly compared with groups without TPCs knockdown, which indicates TM9SF1-induced autophagosome formation is dependent on TPCs knockdown. Therefore, it is of great interest to pursue to discover the detailed functional interaction between TM9SF1 and TPCs on cell autophagy, which is associated with a wide range of diseases including cancer, neurodegenerative diseases, heart diseases, diabetes and infections.
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    Rab GTPase mediated regulation of the autophagic pathway and mTOR signaling in the larval fat body of Drosophila melanogaster
    (2016-07) Ayala-Navarro, Carlos I
    Autophagy is a conserved lysosomal dependent pathway employed by cells during stress conditions as an alternative source of nutrients to maintain cellular homeostasis and promote survival. The pathway is negatively regulated by the mechanistic target of rapamycin (mTOR) and induced by depletion of nutrients. Over the last decade input in the form of vesicular traffic from an array of cellular organelles (e.g. Golgi, ER, endocytic pathway and mitochondria) has been shown to be required for the delivery of proteins, enzymes and lipids during progression of the autophagic pathway. However, how these organelles switch from their constitutive roles to supply the autophagic pathway with proteins and lipids upon induction is not fully understood. In addition the extent to which these cellular organelles modulate autophagosomal growth and mTOR-Insulin signaling remains incompletely understood. The main goal of this thesis was to uncover novel traffic regulators of the Rab GTPase family required for starvation-induced autophagy in Drosophila fat body cells and evaluate their role in mTOR signaling regulation. To this end we carried a reverse screen using RNAi to knockdown 30 of the 33 Drosophila Rab GTPases. We show Rab 2, 7 and 14 GTPases are required for the induction and growth of autophagosomes and autolysosomal function. Rab5 is required for autophagic vesicle induction and growth, endocytosis and lysosomal maturation. Lastly, that Rab6 is required for the sorting of lysosomal hydrolases, autolysosome turnover and the regulation of mTOR signaling via regulation of the insulin receptor localization in fat body cells. Altogether we uncovered novel regulators in the vesicular traffic regulator Rab GTPase family required for autophagy and mTOR-Insulin signaling regulation in Drosophila.
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    Regulation of autophagy by the Unc51 family kinases Atg1 and ADUK in Drosophila melanogaster
    (2015-08) Braden, Christopher
    Regulation of autophagy by the protein complex centered around serine-threonine kinase Atg1 has become well-characterized as a response to nutrient deprivation. However, understanding of how of the Atg1 autophagy induction complex drives autophagy is incomplete. Furthermore, many varied stresses lead to induction of autophagy. While many of these stresses have been linked to canonical autophagy regulation through Atg1, some have been shown to induce autophagy even in its absence. How the autophagic machinery is engaged in the absence of Atg1 remains an open question. Here we describe the induction of autophagy in the absence of Atg1 by a previously uncharacterized Drosophila protein we have named "Another Drosophila Unc-51-like Kinase" (ADUK) after the family of kinases that include ADUK and Atg1. Overexpression of ADUK induces autophagy in WT animals or Atg1 mutants, and GFP-ADUK localizes in punctae at autophagosomes. Interestingly, autophagy induction by ADUK requires Atg1 binding partner Atg13, and, like Atg1, ADUK co-immunoprecipitates with Atg13. Co-expression of Atg1 complex member FIP200 with ADUK enhances autophagy induction and GFP-ADUK punctae formation. Surprisingly, co-expression of Atg13 inhibited both ADUK phenotypes. ADUK mutation results in a reduced post-eclosure lifespan, and inhibits autophagy in response to a complex stressor, DMSO, but not in response to the canonical autophagy stimulus of amino acid deprivation. We conclude that ADUK represents a novel regulator of autophagy, likely engaging the downstream autophagic machinery in a manner similar to Atg1. As an Atg1-independent inducer, ADUK represents a new potential access point for autophagy regulation by non-nutrient stresses.
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    Regulation of developmental timing in Drosophila melanogaster: genetics versus environment
    (2019-07) Pan, Xueyang
    Development of animals involves both an intrinsic program determined by genetics and an adaptive system reacting to environmental variants. In fruit fly Drosophila melanogaster, the juvenile-to-adult transition is largely governed by a neuroendocrine axis in which the PTTH-producing PG neurons and the larval endocrine organ prothoracic gland (PG) play the central role. However, the mechanism underlying the regulation of this neuroendocrine axis is not fully understood. In this thesis two discoveries are made on both the genetic control of the neuroendocrine axis and its response to nutritional stress. Firstly, the author demonstrates that autophagy acts as a nutritionally-regulated gating mechanism which helps ensure productive metamorphosis in Drosophila. Autophagy in the PG is specifically stimulated by nutrient restriction at the early, but not the late third instar larva stage, which inhibits precocious metamorphosis during nutrient restriction in undersized larvae. Induction of autophagy disrupts production of the steroid hormone ecdysone at the time of pupariation not by destruction of hormone biosynthetic capacity, but rather by limiting the availability of the steroid hormone precursor cholesterol in the endocrine cells via a lipophagy mechanism. These findings demonstrate an autophagy mechanism in PG cells that helps shape the nutritional checkpoints and guarantee a successful juvenile-to-adult transition in animals confronting nutritional stress. Secondly, the author shows that Jeb/Alk and Pvf/Pvr pathways function jointly with PTTH/Torso pathway in the PG neuron-PG neuroendocrine axis to control developmental timing in Drosophila. In the two pathways, Jeb and Pvf ligands are expressed in the PG neurons, which activate the Alk and Pvr receptors respectively in the PG. Suppression of the Jeb/Alk or Pvf/Pvr pathway causes developmental timing delay in the larva, which is exacerbated when combined with mutation of ptth. Activation of the pathways rescues the developmental delay caused by ptth mutation, indicating a compensatory effect. These data demonstrate that the Jeb/Alk and Pvf/Pvr pathways are among the previously proposed additional signals from the PG neuron-PG axis which function jointly with the PTTH/Torso pathway to control developmental timing.
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    Studies on the regulatory mechanism of the ULK1 complex in the induction of autophagy
    (2012-10) Cao, Jing
    Autophagy, an evolutionarily-conserved cellular process through which organelles and macromolecules are degraded in the lysosome, is induced under nutrient starvation or other unfavorable growth conditions. Unc51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a key role in the autophagy induction process, but how ULK1 is regulated by cellular signals for induction of autophagy and how ULK1 regulates the downstream processes in autophagy remain poorly understood. ULK1 interacts with Atg13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and Atg101 to form a large protein complex involved in early steps of the autophagy induction process. To better understand the function of the ULK1 complex, my thesis work has sought to identify binding proteins of the complex. Through a yeast two hybrid screen using a human fetal brain cDNA library with Atg13 as bait, a protein named MCF.2 cell line derived transforming sequence-like 2 (MCF2L2) was identified. Through co-immunoprecipitation and in vitro binding assay, MCF2L2 was determined to directly interact with Atg13 via its N-terminal region independently of ULK1. Knockdown of MCF2L2 inhibited the formation of autophagosome and autophagy flux and led to accumulation of p62/sequestosome-1, a protein degraded through autophagy. Knockdown of MCF2L2 also suppressed the aggregation of WD-repeat protein interacting with phosphoinositides-1, an autophagic isolation membrane marker. MCF2L2 contains a putative Rho-guanine nucleotide exchange factor (GEF) domain in the middle and has a sequence similarity to MCF2L and MCF2, the well-known Rho-GEFs. MCF2L2 overexpression induced a moderate increase in the active forms of Rho GTPases and MCF2L2 colocalized with actin related protein 3, the actin nucleation factor that is regulated by Rho GTPases, implying that MCF2L2 potentially contains GEF activity. MCF2L2 knockdown partially suppressed the distribution of Atg9 from trans-golgi network to the cytoplasm in response to starvation, a process that may depend on actin cytoskeleton. Combined, these results suggest that MCF2L2, as a component of the ULK1 complex, might play an important role in mediating signal transduction between the actin cytoskeleton and autophagy induction.
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    Unconventional protein secretion from adipocytes
    (2018-10) Josephrajan, Ajeetha
    Endocrine function of the adipose tissue plays a major role in maintaining energy balance and glucose homeostasis by releasing a large number of bioactive proteins. Any dysfunction of the endocrine function of the adipose tissue caused due to obesity will initiate pathophysiological changes and hasten disease progression. In this thesis, I focus on the secretion of leaderless proteins from the primary cells of the adipose tissue, the adipocytes. This secretion process is called unconventional protein secretion (UPS) and as shown here for the first time, our results indicate that the UPS is highly regulated and a variety of proteins are secreted upon the adipocyte receiving a lipolytic stimuli. To characterize the UPS, we followed the secretion pathway of unconventionally secreted adipocyte fatty acid binding protein (FABP4). FABP4 is one of the majorly expressed protein in mature adipocytes whose intracellular function is lipid storage and trafficking. Increasing evidence indicates that FABP4 has multiple functions extracellularly and is strongly associated with metabolic disease progression. Our results elucidate the regulation and mechanism of UPS/FABP4 secretion pathway. Unraveling the role of UPS proteins in the circulation and integrating them as a systemic response will be central to our understanding of the balance between healthy and unhealthy states. Such a study will be more insightful in predicting metabolic diseases than analyzing different individual marker proteins in the blood stream at a time for various pathologies.