Browsing by Subject "Carrier Proteins"
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Item Biochemical Characterization of Niemann-Pick C: A Disease of Cholesterol Transport(2012-08-15) Infante, Rodney Elwood; Brown, Michael S.; Goldstein, Joseph L.Despite intense scientific interest, the mechanism by which cholesterol is transported between membrane compartments in animal cells remains obscure. One transport pathway begins in lysosomes where cholesterol is liberated from plasma lipoproteins that have entered the cell through receptor-mediated endocytosis. This cholesterol is transported from the lysosome to other cellular membranes to perform structural and regulatory roles. A clue to the mechanism of this cholesterol movement comes from observations in cells from patients with Niemann-Pick Type C (NPC) disease. These individuals accumulate large amounts of cholesterol throughout the body caused by mutations in either one of two genes encoding the lysosomal proteins NPC1 and NPC2. Unlike the membrane protein NPC1, evidence suggests that the soluble protein NPC2 is a cholesterol binding protein. In the course of isolating a cholesterol-homeostasis membrane protein that binds sterols, we encountered NPC1. Using rabbit membranes, an integral membrane protein that bound sterols was isolated with a 14,000-fold purification while maintaining 8% final yield. Mass spectrometry identified the protein to be NPC1. Recombinant human NPC1 was expressed, purified, and confirmed to be a high affinity sterol receptor. NPC1's sterol binding domain was localized to it's N-terminal luminal soluble domain, which can be prepared as a soluble protein of 240 amino acids, NPC1(NTD), that is secreted by cells. The binding properties of NPC1(NTD) binds cholesterol similar to NPC2 with a Kd of ~130nM. Cross-competition studies between purified NPC1(NTD) and the soluble NPC2 protein revealed differences in sterol specificity depicting the different parts of the cholesterol moiety the NPC proteins bind. Finally, an in vitro assay was established to measure transfer of [³H]cholesterol between the two NPC proteins and phosphatidylcholine liposomes. NPC1(NTD) donates or accepts cholesterol from liposomes very slowly, whereas NPC2 acts quickly. NPC2 stimulates the bidirectional transfer of cholesterol between NPC1(NTD) and liposomes. A naturally-occurring human mutant of NPC2 (Pro120Ser) failed to facilitate the movement of cholesterol from NPC1(NTD) to lipid bilayers. These studies directly link both proteins to the cholesterol egress process from lysosomes, explaining how mutations in either protein produce similar clinical phenotypes.Item C. Elegans OMA-1 and OMA-2 are Transcriptional and Translational Repressors of Germline Fate(2009-09-04) Ozkan, Tugba Guven; Lin, RueylingThe germline is a very specialized cell lineage for the proper transmission of genetic material through many generations, to ensure flawless perpetuation of the species and life cycles. The germline lineage is set aside as early as embryogenesis and kept quiescent until germ cells are needed for adult reproduction. During C. elegans germline development global transcription is repressed in specialized, mature diakinetic oocytes of the adult animal and transcription is reactivated as zygotic transcription in the 4-cell stage embryo but only in somatic blastomeres. Global transcription is kept repressed by PIE-1 in germline precursors beginning with 4-cell stage to protect germ lineage from inappropriate somatic differentiation pathways. During my graduate studies, I investigated the redundant roles for two CCCH type RNA binding zinc finger proteins OMA-1 and OMA-2 during C. elegans germline development and early embryogenesis. Previously, OMA proteins were shown to be required for oocyte maturation but they were not assigned any molecular functions. My thesis demonstrates transcriptional repression function of OMA proteins in newly fertilized embryos and translational repression functions during oogenesis. I showed that OMA-1/2 are redundantly required for global transcriptional repression before the onset of zygotic transcription in the 1-cell and 2-cell stages of C. elegans embryos by interacting with and sequestering in the cytoplasm TAF-4, a highly conserved essential basal transcription factor. Nuclear enrichment of TAF-4 requires interaction with another transcription factor TAF-12. OMA-1 competes with TAF-12 to interact with and change subcellular localization of TAF-4, in order to displace TAF-4 away from nuclei and prevent transcriptional initiation. I showed that interaction of OMA-1 and TAF-4 is regulated by MBK-2 phosphorylation at oocyte to embryo transition. My data suggest a model in which MBK-2 phosphorylated embryonic OMA-1 can change TAF-4 subcellular localization only in newly fertilized C. elegans embryos, not during oogenesis. When properly phosphorylated by MBK-2 kinase, ectopic OMA-1 is sufficient to repress transcription in later embryonic stages. Strikingly, reduction of oma-1/2 activities not only results in transcriptional derepression in newly fertilized embryos, but also in later germline blastomeres where wild type OMA-1 is normally absent. I show that OMA-1/2 indirectly repress global transcription in later germline blastomeres by preventing premature degradation of PIE-1 during germline development. OMA proteins protect PIE-1 and other CCCH RNA binding proteins from degradation by repressing zif-1 mRNA translation, the substrate specific binding partner for PIE-1 degradation. A zif-1 3'UTR reporter is repressed in the pachytene and proximal regions of the adult C. elegans germline, and expression of the reporter is activated in the 4-cell embryo only in anterior blastomeres, reciprocal to the PIE-1 expression pattern. I show that zif-1 3'UTR reporter is repressed in the proximal oocytes and in the pachytene region of the germline by OMA-1/2 and GLD-1 respectively. I further showed that zif-1 3'UTR reporter is kept repressed in germline blastomeres of the embryos by POS-1 and SPN-4 and its activation requires anterior cell fate determinants MEX-5/6 during embryogenesis. Contrary to the requirement for MBK-2 phosphorylated OMA-1/2 for embryonic transcriptional repression function, zif-1 3'UTR reporter repression by OMA proteins in the oocytes requires un/hypophosphorylated OMA proteins, the version of OMA-1/2 detected in the oocytes. In summary, my thesis shows that OMA-1/2 are dual function proteins redundantly required for germline development and maintenance of germline identity during oogenesis and embryonic development of C. elegans. OMA proteins are critical for the protection of CCCH type maternal proteins during oocyte development by preventing their premature proteasomal degradation through inhibiting translation of zif-1 mRNA. MBK-2 phosphorylation at the oocyte to embryo transition converts OMA proteins from oocyte translational repressors to embryonic transcriptional repressors. Phosphorylated OMA proteins can interact with TAF-4 in the newly fertilized C. elegans embryos and repress global transcription to prevent premature somatic differentiation during early stages of embryogenesis. OMA proteins protect germline identity at the level of both translational and transcriptional repression during the very critical time points of development to regulate a proper oocyte to embryonic transition.Item Characterization of Alsin and Its Role in IGF-1-Mediated Neuronal Survival(2005-04-29) Topp, Justin David; Horazdovsky, BruceThe transport of proteins between organelles is a highly regulated and complex process that is crucial for many of the functions required for cellular homeostasis. Many distinct proteins are involved in each trafficking step with roles in vesicle formation, budding, movement, and fusion. One class of proteins, the Rab GTPases, is required for docking and fusion of transport vesicles with their target membrane. These proteins are regulated by their state of nucleotide binding, with GTP-bound Rabs thought to provide specificity to transport steps via their interactions with specific effector proteins. While much work has been focused on proteins downstream of Rab GTPases, little is known as to how the activation of these proteins is controlled. This is particularly true of Rab5, the Rab protein required for vesicle fusion at the endosome. Endocytosis of plasma membrane proteins requires Rab5(GTP, and humans possess at least seven proteins (Vps9 family) that are expected to activate Rab5. An intriguing aspect of the Vps9 family of proteins is that they appear to link signal transduction to receptor trafficking via the specific coupling of particular receptors to Rab5-mediated endocytosis. Cell biological, biochemical, and immunohistochemical techniques were employed to characterize one of the Vps9 family proteins named Alsin. Alsin is required for motor neuron maintenance and/or survival, as loss-of-Alsin function results in multiple juvenile-onset neurodegenerative disorders (ALS2, JPLS, IAHSP). It was found here that Alsin is an endosomal protein that activates both Rac1 and Rab5. This protein is present in all of the tissues associated with the aforementioned diseases and intriguingly is upregulated in the cerebellum, an unknown site of pathology for this class of disorders. Alsin was found to couple Rab5 activation specifically to the IGF-1 signal transduction pathway via its regulation of IGF-1 receptor endocytosis. This function of Alsin was shown to be essential for IGF-1-mediated cell survival. These results provide the first characterization of Alsin and identify a novel cause for neurodegeneration.Item A Chemically Induced Colitis Screen Reveals the Necessity for Membrane Traffic in Intestinal Homeostasis(2019-03-21) McAlpine, William Elliott; Winter, Sebastian E.; Beutler, Bruce; Burstein, Ezra; Schmid, Sandra; Scherer, PhilippInflammatory bowel disease is most commonly a complex disorder caused by the interaction of environmental and genetic aberrations. Under normal conditions, a genetic program actively prevents inflammatory bowel disease, preventing invasion of microbes without permitting severe inflammation of the gut. To identify genes that maintain this balance, we performed a sensitized screen of 49,420 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires, bearing 104,658 coding/splicing mutations. We induced mild mucosal damage in these mice by orally administering dextran sodium sulfate (DSS) and found mutations that led to diarrhea and weight loss under these conditions. Causative mutations were mapped concurrently with screening using an automated mapping procedure. Among 114 DSS phenotypes identified and mapped, 36 have been validated by CRISPR/Cas9 targeting. Three vesicle trafficking genes, Myo1d, Smcr8, and Tvp23b, were selected for mechanistic evaluation. MYO1D is a class I myosin that binds both actin and lipid. MYO1D localizes to the basolateral membrane of enterocytes and functions in the intestinal epithelium to protect against colitis. SMCR8, along with C9ORF72 and WDR41, is a member of a tripartite complex that functions as a guanine exchange factor. SMCR8 localizes to the lysosome, and its absence results in perturbations to endocytic and phagocytic pathways. Hyperactivation of endosomal Toll-like receptors in Smcr8-/- mice causes spontaneous inflammation, and hyperactivation of multiple pathways contributes to DSS susceptibility. TVP23B is a trans-Golgi protein that binds YIPF6. Both TVP23B and YIPF6 are necessary for the formation of secretory granules in goblet and Paneth cells of the intestinal epithelium. These studies reveal non-redundant molecules required for the return of normal physiologic balance within the intestine after DSS insult.Item Cholesterol Accessibility in Membranes(2019-03-19) Endapally, Shreya; Alto, Neal; Radhakrishnan, Arun; Rizo-Rey, José; Blount, PaulCholesterol levels in mammalian cells are tightly regulated to lie within narrow limits. This regulation is achieved by employing multiple feedback mechanisms to regulate both synthesis and uptake of cholesterol. Most of a cell's cholesterol (~80 % of total) is in the plasma membrane (PM), but the protein machinery that senses and regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell's cholesterol. A carefully regulated lipid transport pathway between PM and ER allows cholesterol sensors in ER to monitor the cholesterol content of cholesterol-rich PM. This transport depends on the interactions of cholesterol with various phospholipids that control its accessibility for transport to the ER. Cholesterol in PM is organized into three different pools. One pool is accessible for transport to the ER, a second pool is sequestered by sphingomyelin (SM) and can be released by treatment with sphingomyelinase, and a third pool remains sequestered even after sphingomyelinase treatment. Here, I describe our work in developing and characterizing tools to study these different pools. The three pools were identified using bacterial toxins called cholesterol dependent cytolysins (CDCs) which selectively bind to the accessible pool of cholesterol. One example of these toxins is Anthrolysin O (ALO). To better understand the dynamics of accessible cholesterol sensing at a molecular level, we developed a stable construct of the cholesterol sensing domain of ALO (ALOD4) and performed NMR and other biophysical studies using cholesterol containing model membranes. We were able to identify residues that are significantly affected by the interaction of ALOD4 to membranes. We also developed a highly specific sensor for the SM-sequestered pool of cholesterol. This sensor is derived from a fungal toxin, Ostreolysin A (OlyA). Using X-ray crystallography, we studied the interaction of OlyA with SM and cholesterol at the atomic level. This structural analysis combined with detailed mutagenesis led us to a single point mutation in OlyA that abolishes its cholesterol specificity while retaining SM specificity. Comparing the X-ray structures of these two versions of lipid bound OlyA combined with ligand docking simulations revealed two distinct conformations of SM: one in complex with cholesterol and one free from cholesterol. Studies in live cells using OlyA and ALOD4 show that the pool of SM/cholesterol complexes in plasma membrane is maintained at a constant level across a large range of cholesterol concentrations. The development of new tools for specific forms of cholesterol (ALOD4 and OlyA) has allowed us to evaluate long-standing hypotheses regarding lipid organization in PMs and has also shed new light on lipid dynamics in the context of cellular signaling.Item Discovery and Characterization of the Polycomb Repressive Complex 1 of C. Elegans(2009-06-17) Karakuzu, Ozgur; Cameron, Scottunc-3 encodes the Caenorhabditis elegans homolog of the Olf-1/Early B cell factor family of transcription factors, which in vertebrates regulate development and differentiation of B lymphocytes, adipocytes, and cells of the nervous system. In the first chapter I analyze the role of unc-3 in determining the fates of neurons in ventral nerve cord (VNC). unc-3 mutants are uncoordinated in locomotion. I show that unc-3 represses a VC-like motor neuron program in the VA and VB motor neurons, which in wild-type animals control backwards and forwards locomotion, respectively. Our lab identified a physical interaction between UNC-3 and the C2H2 zinc finger transcription factor PAG-3, the mammalian homologs of which are coexpressed in olfactory epithelium and hematopoietic cells. Our data explain the locomotory defects of unc-3 mutants and suggest that interactions between unc-3 and pag-3 homologs in other species may be functionally important. The second chapter of the thesis is about the analysis of MIG-32 a RING protein similar to some Polycomb proteins that were identified in a yeast two hybrid screen with UNC-3 as bait. The Polycomb repression complex 2 (PRC2) methylates histone H3 lysine 27 at target genes to modify gene expression, and this mark is recognized by PRC1, which ubiquitylates histone H2A. In Caenorhabditis elegans, a complex of the MES-2, MES-3, and MES-6 proteins is functionally analogous to the PRC2 complex, but the functional analog of PRC1, and indeed whether C. elegans has such a complex, has been unclear. I describe here that MIG-32 is a homolog of BMI-1, a core component of PRC1. I also identify SPAT-3A as a homolog of Ring1B, a partner protein of BMI-1 in the PRC1 core complex. Mig-32 and spat-3 mutants have some defects that overlap with those of mes mutants. However, unlike the mes mutants, mig-32 and spat-3 mutants are fertile, despite lacking apparent H2A ubiquitylation. Migration and axon guidance of specific neurons were defective in mig-32 and spat-3 mutants. Our data suggest that mig-32 and spat-3 encode core components of a PRC1-like complex in C. elegans.Item Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item The Hydrophobic Handoff Between NPC2 and the N-Terminal Domain of NPC1 in the Export of Cholesterol from Lysosomes(2013-05-31) Wang, Michael Leechun; Brown, Michael S.; Goldstein, Joseph L.; Thomas, Philip J.; Roth, Michael G.; Hofmann, Sandra L.; Liang, GuoshengLow density lipoproteins (LDL) and related plasma lipoproteins deliver cholesterol to cells by receptor-mediated endocytosis. The lipoprotein is degraded in late endosomes and lysosomes where its cholesterol is released. Egress of cholesterol from late endosomes and lysosomes (hereafter referred to as lysosomes) requires two proteins: Niemann-Pick C2 (NPC2), a soluble protein of 132 amino acids; and NPC1, an intrinsic membrane protein of 1278 amino acids and 13 postulated membrane-spanning helices that span the lysosomal membrane. Recessive loss-of-function mutations in either NPC2 or NPC1 produce NPC disease, which causes death in childhood owing to cholesterol accumulation in lysosomes of liver, brain, and lung. Consistent with their cholesterol export role, NPC2 and NPC1 both bind cholesterol. The cholesterol binding site on NPC1 is located in the NH2-terminal domain (NTD), which projects into the lysosomal lumen. This domain, designated NPC1(NTD), can be expressed in vitro as a soluble protein of 240 amino acids that retains cholesterol binding activity. This thesis studies NPC2 and NPC1(NTD) in detail as summarized below. Two major differences exist between the cholesterol binding of NPC2 and NPC1(NTD). 1) Competitive binding studies and crystal structures indicate that the two proteins bind cholesterol in opposite orientations. NPC2 binds the iso-octyl side chain, leaving the 3ß hydroxyl exposed, whereas NPC1 binds the 3ß-hydroxyl, leaving the side chain partially exposed. 2) Kinetic studies of cholesterol binding reveal that NPC2 binds and releases cholesterol rapidly (half-time < 2 min at 4oC), while NPC1(NTD) binds cholesterol very slowly (half-time > 2 hr at 4oC). Its rapid cholesterol binding allows NPC2 to transfer cholesterol to and from liposomes. Unlike NPC2, NPC1(NTD) cannot rapidly transfer its bound cholesterol to liposomes. However, NPC1(NTD) can accomplish this delivery when NPC2 is present. Furthermore, cholesterol binding to NPC1(NTD) is accelerated by >15-fold when the sterol is first bound to NPC2 and then transferred to NPC1(NTD). These data led us to advance a model in which NPC2 can mediate bi-directional transfer of cholesterol to or from NPC1(NTD). In cells, we envision that NPC2 accepts cholesterol in the lysosomal lumen and transports it to membrane-bound NPC1, thus accounting for the requirement for both proteins for lysosomal cholesterol export. Amino acid residues important or binding or transfer of cholesterol on NPC2 and NPC1(NTD) were identified through alanine scan mutagenesis. For both NPC2 and NPC1(NTD), residues that decreased binding mapped to areas surrounding the binding pockets on the crystal structures; residues that decreased transfer, but not binding, mapped to discrete surface patches near the opening of the binding pockets. These surface patches may be sites where the two proteins interact to transfer cholesterol. The most severe mutations disrupting binding were P120S for NPC2 and P202A/F203A for NPC1(NTD); and those that disrupted transfer were V81D for NPC2 and L175Q/L176Q for NPC1(NTD). Furthermore, the functional significance of both the binding and transfer of cholesterol by NPC2 and NPC1(NTD) in the egress of cholesterol from lysosomes was confirmed. The above binding- or transfer-defective mutants of NPC2 and NPC1 were unable to rescue LDL-stimulated cholesteryl ester synthesis in NPC2 or NPC1-deficient cells, respectively, in contrast to wild-type NPC2 and NPC1. With these data, we envision that NPC2 binds cholesterol the instant that it is released from LDL, either as the free sterol or after cleavage of lipoprotein-derived cholesteryl esters by lysosomal acid lipase. This binding would prevent cholesterol from crystallizing in the lysosomal lumen. According to the model, NPC2 can transfer its bound cholesterol to NPC1(NTD) directly, thus avoiding the necessity for the insoluble cholesterol to transit the water phase. This transfer of cholesterol from NPC2 to NPC1(NTD) has a special functional relevance in light of the near-absolute insolubility of cholesterol in water, and we have named this process a "hydrophobic handoff."Item The Impact of Pleiotrophin on Breast Cancer Progression(2019-03-12) Sorrelle, Noah Bruce; Cobb, Melanie H.; Brekken, Rolf A.; Scherer, Philipp; Sternweis, Paul C.Breast cancer is the most frequent type of cancer, despite being largely restricted to women. Over the past few decades, cancer biologists have made great strides in understanding the factors that drive breast cancer tumorigenesis and progression. However, the significance of many factors, such as Pleiotrophin, remain uncharacterized. Pleiotrophin (Ptn), a neurite outgrowth factor and a heparin-binding cytokine, is reportedly expressed in many types of cancer, including breast cancer. Despite being identified as a secreted factor produced by breast cancer cells in 1991, its functional significance in breast cancer is uncertain. Previous studies into this question were limited by available tools for specifically perturbing or knocking out Ptn genetically. Further, they were also limited in scope, focusing only on select characteristics of the tumor progression and the microenvironment. Curiously, research into Ptn's impact in breast cancer came to a halt in 2007, leaving the very question of Ptn's functional significance unanswered. Using pharmacologic and genetic methods, I tested the impact of Ptn perturbation in multiple preclinical models of breast cancer. Ptn perturbation only impacted primary tumor growth in a single model. In contrast, Ptn perturbation resulted in reduced pulmonary metastatic burden in every model tested thus far. This effect does not appear to be due to Ptn's direct impact on cancer cell phenotype, growth, or migration. Anti-Ptn therapy did not affect epithelial-tomesenchymal transition of cancer cells, nor did Ptn directly promote cancer cell proliferation or migration in vitro. These results suggest that Ptn's effects are through changes in the tumor microenvironment. By immunohistochemistry and flow cytometry, I observed that there was less neutrophil infiltration and macrophage/metastatic lesion coupling in vivo. This result was interesting as both neutrophils and macrophages are implicated in promoting pulmonary metastasis in preclinical models of breast cancer. Using a cytokine/chemokine array, I observed that Ptn perturbation resulted in the reduction of macrophage and neutrophil chemokines, including MCP-1 and CXCL5, respectively. In contrast to previous reports, Ptn perturbation did not result in changes in angiogenesis, epithelial-to-mesenchymal transition, or markers of cell proliferation or apoptosis. Some of these reported activities may have resulted from off-target effects of the tools used at the time. Overall, the experimental strategies and specificity of the tools used in my thesis work have provided more accurate insights into the activity of Ptn. Additionally, the results suggest that Ptn is a potent driver of pulmonary metastasis in breast cancer and that targeting Ptn may an effective therapeutic strategy to treat metastatic breast cancer.Item In Pursuit of a Molecular Fountain of Youth: The Identification and Characterization of Lifespan Regulators in Drosophila(2012-07-20) Stenesen, Drew Stanness; Graff, Jonathan M.Over the past century, average human lifespan has experienced steady increase despite lack of substantial intervention or understanding of the aging process. In fact, many organisms have the latent potential to live much longer than they normally do. This indicates lifespan determination is an active process subject to regulation. Components of this impending longevity are beginning to unravel through dietary and genetic studies in model systems. To date, several pathways indicate human lifespan extension through direct molecular intervention may be feasible, however, important limitations persist. A common thread among these conserved lifespan regulators is metabolism. Therefore, further insight into lifespan extending mechanisms may lie within tissues governing important metabolic processes. Here we describe a multi-tiered, strategy to identify Drosophila melanogaster mutants with extended lifespan based upon enrichment for insertions in genes that are expressed in metabolic tissues. Our results indicate metabolically relevant tissues are a rich source of genetic longevity regulation. We identified a regulator of G protein signaling (RGS) domain containing sorting nexin, termed snazarus (sorting nexin lazarus, snz). Flies with insertions into the 5' untranslated region of snz live up to twice as long as controls. Transgenic expression of UAS-Snz from the snz Gal4 enhancer trap insertion, active in metabolic tissues, rescued lifespan extension. Notably, old snz mutant flies remain active and fertile indicating that snz mutants have prolonged youthfulness, a goal of aging research. Since mammals have snz-related genes, it is possible that the functions of the snz family may be conserved to humans. Next, we identified the two key adenosine monophosphate (AMP) biosynthetic pathways as regulators of Drosophila longevity. We found that heterozygous mutation of anabolic components of the de novo as well as the salvage AMP biosynthesis pathways extend lifespan. These pathway mutations, and caloric restriction, increased adenosine mono- and diphosphate to adenosine triphosphate (ATP) ratios. Consistent with the altered ratios, lifespan extension was dependent on functional adenosine monophosphate-activated protein kinase (AMPK). Supplementing the diets of adult mutants with adenine restored adenosine nucleotide ratios and rescued lifespan extension. These data establish de novo and salvage AMP biosynthesis as determinants of adult lifespan. The dosage sensitivity and enzymatic nature of de novo and salvage AMP biosynthesis, and the conserved aspects of adenosine nucleotide derivatives and lifespan extension, indicate that these pathways are potentially amendable drug targets worth continued exploration.Item KU70 Binding Protein 5 (KUB5), A Novel Factor in DNA Double Strand Break Repair and Radio-Resistance in Human Breast Cancer(2011-02-01) Rommel, Amy Ann; Boothman, David A.DNA double strand breaks (DSBs) are considered both mutagenic and carcinogenic if left un-repaired resulting in genomic instability and ultimately cancer. There are two main pathways for DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). Defects in DSB repair have already been associated with breast cancer formation and increased breast cancer risk. Breast cancer susceptibility genes, BRCA1 and BRCA2 are largely thought to be involved with HR while LIG4, XRCC4, and Ku70 are linked to NHEJ. Deficiencies in any one of these genes can predispose individuals to breast cancer. In addition to predisposition to breast cancer, altered DNA repair processes can influence chemo- and radiotherapy efficacy by creating resistance to therapy. To study NHEJ further, our laboratory has identified a novel Ku70 binding protein #5 (KUB5) by a yeast two-hybrid screen using Ku70 as bait. Loss of RTT103, a putative yeast homolog of KUB5, resulted in increased sensitivity to IR, similar to that observed in hdf1-deletion yeast, the yeast homolog of Ku70. Results also show that RTT103-deletion yeast are deficient in repairing blunt and non-compatible DNA ends and re-expression of hKub5 can correct the IR-sensitivity and DNA repair deficiency of these deficient yeast demonstrating a strong functional model for human KUB5 function in yeast. Analyses of breast cancer cell lines for their KUB5 protein expression yielded a strong correlation between KUB5 protein level and sensitivity to DNA damage. These data strongly suggests that KUB5 is a novel repair factor involved in NHEJ and endogenous over-expression of KUB5 plays a role in chemotherapeutic and/or radio-therapeutic resistance via increasing the capacity to facilitate NHEJ repair of DSBs in breast cancer cells.Item A Novel Role for Odorant Binding Proteins in Deactivation of Drosophila Olfactory Neurons(2019-07-29) Scheuermann, Elizabeth Anne; Meeks, Julian P.; Smith, Dean P.; Krämer, Helmut; Terman, Jonathan R.In insects, odorant binding proteins are a large and diverse group of low molecular weight proteins secreted into the fluid bathing olfactory and gustatory dendrites. The best-characterized OBP, known as LUSH, is required in Drosophila melanogaster for the detection of physiological levels of the male-specific pheromone cVA. While LUSH acts as a sensitizing factor for pheromone detection, the role of other OBPs encoded in the Drosophila genome is largely unknown. In an effort to characterize members of this family, I used CRISPR-Cas9 to generate and characterize a deletion of two genes encoding the homologous OBPs OS-E and OS-F. These OBPs are nearly 70% identical and their expression is restricted to a small set of antennal chemosensory sensilla. Electrophysiological analysis of the olfactory neurons within these sensilla revealed no major difference in odorant sensitivity or specificity in the mutants but did reveal a striking deactivation defect to a subset of odorants. Surprisingly, other odorants detected by the same receptor are differentially affected by the absence of OS-E and OS-F, revealing an odorant-specific role for these OBPs in deactivation kinetics. Activation kinetics remain normal for the affected odorants in mutants. Genomic rescue experiments revealed that OS-E and OS-F are also functionally redundant, as either OBP is sufficient to revert the mutant phenotype. My findings reveal a new role for OBPs in deactivation of olfactory neurons and expand our understanding of the range of OBP functions.Item On Cholesterol Transport Between Membranes(August 2021) Trinh, Michael Nguyen; Mendell, Joshua T.; Chen, Zhijian J.; Abrams, John M.; Brown, Michael S.; Goldstein, Joseph L.The studies described in this dissertation focus on investigation of the pathways for transport of cholesterol from one organelle to another in animal cells. Cells have evolved elaborate transport mechanisms to assure an optimum cholesterol content within their membranes. Dysregulation of cholesterol transport causes common diseases, including atherosclerosis. The major source of cellular cholesterol comes from Low Density Lipoprotein (LDL). When plasma membranes are low in cholesterol, cells produce LDL receptors which bind LDL and mediate its uptake by endocytosis and its delivery to lysosomes. Within lysosomes the cholesteryl esters of LDL are hydrolyzed. The free cholesterol binds to a soluble lysosomal protein called Niemann Pick C2 (NPC2) which delivers it to a membrane-embedded protein called NPC1 which inserts the cholesterol into the lysosome membrane. From there the cholesterol moves to the plasma membrane (PM) through a pathway that is unknown. When the PM becomes saturated with cholesterol, any excess is transported to the endoplasmic reticulum (ER) to repress production of LDL receptors and to be stored in lipid droplets. The work described here 1) showed that triazole antifungal drugs inhibit lysosomal cholesterol export by binding to the membrane domain of NPC1 2) used itraconazole to solve the crystal structure of NPC1 at 3.3Å, 3) revealed that cholesterol is transported out of lysosomes through interactions between two or more NPC1 molecules, and 4) utilized CRISPR-Cas9 whole-genome knockout screens to identify all the genes involved in the transport and uptake of LDL cholesterol. From these screens in the latter study, we discovered that a specific phospholipid, phosphatidylserine (PS), is required for PM-to-ER cholesterol transport. These studies provide supporting evidence towards a vision of one-way directional transport of LDL-derived cholesterol from lysosomes to the PM to the ER.Item Post-Transcriptional Regulation of Maternal mRNA Shapes Early C. Elegans Embryogenesis(2014-03-17) Burleson, Marieke Oldenbroek; Buszczak, Michael; Abrams, John M.; Cleaver, Ondine; Hobbs, Helen H.Much of early embryogenesis is controlled through complex networks comprised of maternally provided factors. Oocytes are packed with protein and RNA that are ready to spring into action after fertilization to guide early embryonic development. The regulation of maternally provided factors is therefore critical and is a fundamental goal of developmental biology. During my studies, I investigated how two maternally provided mRNAs, zif-1 and mom-2, are regulated post-transcriptionally through their 3’ untranslated region (3’ UTR) to ensure proper spatio-temporal protein expression. I discovered that seven RNA binding proteins bind directly to the zif-1 3’ UTR in a combinatorial fashion thereby ensuring that zif-1 is only translated in somatic blastomeres, beginning at the four cell stage embryo. Interestingly a similar set of RNA binding proteins (nine total) regulate the spatio-temporal expression of mom-2 in a similar fashion despite the fact that mom-2 has a reciprocal expression pattern when compared to zif-1. My studies on zif-1 and mom-2 regulation indicate that a “code” is embedded within the 3’ UTR of mRNAs to mediate translational regulation. The precise combination of RNA binding proteins present in a particular cell at a particular time, each with the intrinsic capability of binding to regulatory sequences contained in this “code”, determines when and where mRNAs get translated. I also investigated mechanisms by which maternal mRNAs get degraded. Zygotic transcription activation is often linked to maternal mRNA degradation, which I showed to be the case in C. elegans embryos. Specifically, I discovered a gene termed vet-5 that is first transcribed in the somatic blastomeres of the four-cell embryo and is sufficient to degrade at least several maternal mRNAs when provided exogenously as dsRNA. vet-5 maps to a highly repetitive locus and has been shown to be a target of siRNA production. Consistent with vet-5 derived siRNA production I found that the siRNA pathway is, at least partly, required for the degradation of maternal mRNAs and that removing components of the siRNA pathway affects vet-5 expression. Therefore, I hypothesize that siRNAs could be produced from the vet-5 locus that target maternally provided mRNAs for degradation.Item Purification of Native and Recombinant NPC1(2008-12-23) Dale, Jarrod Donald; Goldstein, Joseph L.The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoproteinderived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. We encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. Described here is the initial purification of rabbit NPC1 using a classical biochemical approach and an analysis of the sterol binding properties of native and recombinant NPC1. Our purification yielded a membrane-bound 25-HC-binding protein which was purified more than 14,000-fold from rabbit liver membranes. This protein was identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Initial characterization of the sterol binding properties showed specific binding for 25-HC; however, we were unable to demonstrate significant binding of NPC1 to cholesterol using our current experimental conditions. The availability of assays to measure NPC1 sterol binding in vitro may further the understanding of intracellular sterol transport.Item Regulation of Blood Vessel Development via Rho Family GTPase Signaling(2016-11-28) Barry, David Michael; Cobb, Melanie H.; Carroll, Thomas J.; Ross, Elliott M.; Cleaver, OndineThe Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain unclear. Here, we assess the cellular mechanisms by which Rho family GTPase proteins and their regulators regulate mammalian vascular morphogenesis and maintenance. First we assessed the role of the Rho GTPase Cdc42 during vasculogenesis and angiogenesis. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. Subsequently, Rho GTPase regulatory proteins were further investigated to decipher their role during vascular development. Rasip1 was identified as a promising anti-angiogenesis candidate, which is required for the formation of continuous vascular lumens in growing vessels. Molecular bottlenecks were elucidated during vessel formation by dissecting the cellular events that require Rasip1. We show that Rasip1 controls different GTPase signaling pathways that converge upon the actomyosin contractility machinery. We find that different pools of NMII, downstream of Rasip1, control two different processes in endothelial cells: 1. NMII mediates the removal of pre-apical membrane adhesions to form a lumen. 2. NMII then restrains apical membrane expansion, thereby limiting lumen diameter during vessel growth. In the first process, Rasip1 promotes actin contractility via Cdc42 and Pak4 along ribbons of adhesions at the center of EC cords, causing adhesions to clear from the pre-apical membrane. This allows opening of lumens. Subsequently, Rasip1 inhibits NMII and membrane contractility via RhoA suppression to allow regulated lumen expansion. These novel and distinct spatiotemporal molecular and cellular events define the stepwise process of blood vessel morphogenesis and differentiation.Item Regulation of WASP in Actin Signaling: From Angstroms to Microns(2011-08-10) Cheng, Hui-Chun; Rosen, Michael K.Wiskott-Aldrich syndrome protein (WASP) regulates membrane-attached force generation by promoting actin polymerization, which is key to cell motility and immune responses. Mutations in WASP lead to Wiskott-Aldrich syndrome and neutropenia. My graduate research was focused on allosteric regulation of WASP by an Enterohaemorrhagic E. coli (EHEC) effector EspFU. This study leads to novel mechanisms of WASP regulation and also sheds light on how cells conduct μm-scale assembly by orchestrating nm-scale proteins spatially and temporally. EHEC hijacks the host cytoskeleton during infection to form actin-rich ‘pedestals’ beneath the invading bacteria. The multiple repeat-containing effector EspFU stimulates pedestal formation by activating WASP, which is itself autoinhibited through interactions between a regulatory GTPase binding domain (GBD) and an activity bearing VCA region. First, I investigated how a single repeat fragment (1R) from EspFU relieves WASP autoinhibition. By determining the solution structure of a complex between the WASP GBD and 1R, I showed that EspFU binds to WASP by mimicking the C region of the VCA domain. 1R has a higher affinity toward the GBD than does VCA, allowing it to displace the VCA and activate WASP. Next, I examined the role of multiple repeats of EspFU. Surprisingly, a two-repeat fragment is much more potent in stimulating WASP-dependent actin polymerization than a single repeat at the same total repeat concentration. My colleagues and I showed that the inter-repeat cooperativity of EspFU originates from its ability to engage two active WASP molecules. Such dimers have much higher affinity (~100-fold) than monomers for the actin nucleation factor, the Arp2/3 complex. The combined mechanism enables EHEC to dominate the eukaryotic cytoskeletal machinery. These studies culminated in discovery of the hierarchical regulation of WASP proteins: allosteric activators release autoinhibition, and dimerization/oligomerization of active WASP molecules further enhances activity in promoting actin assembly by the Arp2/3 complex. Multivalency is widespread from extracellular sugar binding proteins to transmembrane receptors to cytoplasmic adapters to nuclear chromatin. Multivalent interactions play a fundamental but incompletely understood role in biology. To assess its roles in signal transduction, my colleagues and I utilized a battery of biochemical and biophysical tools to study a model system, where each multivalent molecule harbors identical modules, and a natural system, where each multivalent molecule consists of homologous modules. In the model system, I observed μm-scale droplet-like protein assembly (phase separation) upon mixing multivalent proteins above a critical concentration. The critical concentration correlates with the valency and individual module binding affinity. In the natural system, the multivalent signaling network consisting of WASP, Nck and Nephrin is necessary to maintain the structural and functional integrity of glomeruli in the kidney. Upon phosphorylation, three phosphotyrosine motifs in the cytoplasmic domain of a transmembrane scaffolding protein Nephrin recruit the C terminal SH2 domain of Nck, whose three SH3 domains interact with multiple proline-rich motifs of WASP. We also observed phase transitions in this system and found that the phase boundary position is highly dependent on the degree of nephrin phosphorylation, suggesting that kinases could induce phase transitions to remodel cellular structure. Furthermore, the phase transition here correlates with a sharp transition in the ability of WASP to stimulate actin assembly by the Arp2/3 complex. Together, this work suggests that cells may exploit multivalency as one of means of regulating the spatial organization and biochemical activity of signaling molecules in response to the environment.Item Role for NIP45 in Telomere Recruitment to PML Bodies in ALT Cancer Cells(2013-05-14) Farley, Demetra Dannielle; Cobb, Melanie H.; Yu, Hongtao; Corey, David R.; Scaglioni, Pier PaoloTelomere length maintenance is critical for continued cell proliferation. The SMC5/6 complex, required for double-strand break (DSB) repair in both yeast and humans, has been implicated in the maintenance of telomere length in certain cancer cells. In the absence of active telomerase, SMC5/6 complex-dependent homologous recombination is utilized to maintain telomere length at PML bodies, a mechanism referred to as alternative lengthening of telomeres (ALT). Sumoylation of several telomere-binding proteins is required for the localization of telomeres to PML bodies in G2 phase cells (APBs). We demonstrate that NIP45, a SUMO-like domain (SLD) containing protein, also affects telomere targeting in ALT cells. Loss of endogenous NIP45 protein results in decreased localization of telomeres to PML bodies in a manner independent of the SMC5/6 complex. NIP45 stimulates telomere binding protein sumoylation, as knockdown of the NIP45 protein negatively affects their sumoylation. Importantly, the NIP45 C-terminal SUMO-like domain (SLD2) is sufficient to rescue both APB formation and telomere-binding-protein sumoylation. NIP45 localizes to PML bodies, but not telomeres, in log phase cells, yet interacts efficiently with TIN2, a sumoylatable telomere binding protein. Additionally, a fragment of NIP45 containing the functional SLD2 domain is sufficient to maintain TIN2 binding. We predict, then, that NIP45 might act to recruit telomeres to the PML bodies via its interaction with TIN2, ultimately allowing for SMC5/6 complex-dependent telomere maintenance in G2 phase cells. In keeping with this hypothesis, loss of endogenous TIN2 protein also negatively affects localization of telomeres to PML bodies, even in the presence of NIP45, supporting a requirement for the TIN2-NIP45 interaction in telomere localization to PML bodies. Through this work, we have defined a role for the NIP45 protein in ALT cancer cell telomere length maintenance, further detailing the mechanism by which telomerase-negative cancer subtypes achieve unlimited replicative potential.Item The Role of Homer Scaffolding to Metabotropic Glutamate Receptor 5 in the Mouse Models of Neurodevelopement Disorders(2014-04-16) Collins, Katie Anne; Kavalali, Ege T.; Albanesi, Joseph P.; Parada, Luis F.; Huber, Kimberly M.Autism is a neurological disorder characterized by repetetive behaviors, social anxiety and verbal and non-verbal communication. Fragile X Syndrome (FXS) is the most common genetic cause of autism and inheritable form of intellectual disability. FXS is caused by the transcriptional silencing of the Fmr1 gene, which encodes for the Fragile X Mental Retardation Protein (FMRP), which is a Ribonucleic acid (RNA) binding protein. FMRP binds to messenger RNA (mRNA) and suppresses their translation. FMRP regulates hundreds of mRNAs, making it a complex disease with several possible dysfunctions causing the many symptoms, like audiogenic seizures or hypersensitivity. While there are several studies which rescue phenotypes, there is little known about what causes the abnormalities, and if it is possible to replicate the symptoms with a single genetic manipulation. There is also little know about common links between different genetic causes of autism. In this study, I manipulate the interaction between metabotropic glutamate receptor 5 (mGluR5) and Homer, and report how these interactions are important in causing some of the phenotypes in FXS. By rescuing disrupted mGluR5-Homer interactions with a Homer 1a knock out I can rescue several phenotypes, and by disrupting mGluR5-Homer interactions with an mGluR5 knock-in mouse that is mutated so it cannot bind to Homer, I was able to mimic them. I was able to rescue/mimic increased basal translation, altered mGluR-signaling, increased neocortical excitability, decreased anxiety, and partial rescue/mimic audiogenic seizures seen in Fragile X mice. However, I was unable to rescue/mimic the enhanced mGluR-LTD. In this study, I also report how the disrupted mGluR5-Homer interactions are caused by Homer being phosphorylation by CaMKIIα. CaMKIIα is an FMRP target and elevated in the Fmr1 KO mice, causing increased phosphorylation of Homer. Inhibiting CaMKIIα, rescues mGluR5-Homer interactions, basal protein synthesis rates and increased neocortical excitability. Lastly, I report how in PTEN conditional KO (cKO) mice, another autism model, also have disrupted mGluR5-Homer interactions. The PTEN cKO mice have increased neocortical excitability, which can be rescued by inhibiting mGluR5. This suggests a common mGluR5 dysfunction in multiple autism models, which could lead to a common treatment.Item The Role of KIBRA in Synaptic Plasticity Across Age(2021-05-01T05:00:00.000Z) Mendoza, Matthew Lee; Meeks, Julian P.; Huber, Kimberly M.; Green, Carla B.; Terman, Jonathan R.; Volk, Lenora J.Over the last four decades, neurobiology has gained valuable insight into the cellular and molecular mechanisms of learning and memory. However, a complete understanding of how we learn and remember information remains at the frontier of neuroscience research. In particular, the molecular bases for age-dependent changes in our capacity to learn and remember are poorly understood. Identifying the neural basis of age-dependent changes in learning and memory will not only provide crucial insights into the pathological mechanisms underlying progressive neurological disorders but also guide neurodevelopmentally informed educational strategies and legal policies. Synaptic plasticity, expressed as persistent increases (long-term potentiation, LTP) or decreases (long-term depression, LTD) in synaptic strength is thought to be a key cellular mechanism underlying cognitive functions such as learning and memory. AMPA-type glutamate receptors mediate the vast majority of fast-excitatory synaptic transmission in the central nervous system, and dynamic AMPA receptor trafficking is critical for many forms of synaptic plasticity. The coordinated movement of AMPA receptors into and out of a synapse is regulated by interactions with multiple proteins including the synaptic scaffold KIBRA (Kidney and Brain Protein). Previous evidence indicates that KIBRA and it's respective binding partners are associated with age-emergent neurological diseases such as Tourette, Schizophrenia, Alzheimer's, and Autism Spectrum Disorders. While there is a growing body of human literature implicating KIBRA in learning and memory, KIBRA's molecular function and contribution to cognitive maturation remains poorly understood. Therefore, this dissertation was designed to focus on the role of KIBRA in synaptic plasticity and AMPA receptor trafficking across the juvenile and adult brain. In Chapter 1, I review the pertinent literature to frame the overall trajectory of this dissertation. Next, in Chapter 2, using novel inducible and conditional KIBRA knock mice, I show that KIBRA acutely influences hippocampal LTP selectively in the adult brain, but not the juvenile brain. These adult-specific deficits in LTP were associated with a reduction in the basal and activity-dependent expression of AMPA receptors and AMPA receptor complex interactors. In Chapter 3, I examine KIBRA's role in LTD. Contrary to published results in conventional KIBRA KO mice on a hybrid C57Bl6N/FVB background, we show that acute manipulation of KIBRA on a C57Bl6N background does not influence hippocampal LTD. Lastly, I show that acute reduction of KIBRA influences GluA2 phosphorylation at S880, which might restrict the recycling of internalized AMPA receptors. Taken together, my data suggest that KIBRA preferentially influences LTP as opposed to LTD. KIBRA's role in LTP is selective to the adult hippocampus and loss of KIBRA reduces the expression and trafficking of AMPA receptors. In Chapter 4, I discuss the implication of this work and layout future directions.