Browsing by Subject "Endoplasmic Reticulum"
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Item Ceramide-Induced Alternative Translocation of TM4SF20(2015-10-29) Lee, Ching En; Pfeiffer, Julie K.; McKnight, Steven L.; Kahn, Jeffrey; Nijhawan, Deepak; Ye, JinThe polytopic membrane protein TM4SF20 (transmembrane 4 L6 family 20) is a protein containing four transmembrane helices that inhibits the Regulated Intramembrane Proteolysis (RIP) of the transcriptional factor CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor. CREB3L1 RIP is induced by several stimuli: ER stress, viral infections, the chemotherapeutic drug, doxorubicin, and the sphingolipid, ceramide. Additionally, TGF-β (transforming growth factor-β), a cytokine known to stimulate collagen production, induces the proteolytic activation of CREB3L1 in human A549 cells through inhibition of TM4SF20 expression, which normally inhibits RIP of CREB3L1. We also find that the TM4SF20 regulation of CREB3L1 RIP is regulated by ceramide. In this study we find that ceramide can regulate the ability of first transmembrane domain of TM4SF20 to determine its orientation in the membrane. Under normal conditions, TM4SF20 is synthesized as a protein that inhibits the cleavage of CREB3L1 when TRAM2 (translocation associated membrane protein 2) is associated with the ER translocon. Excess ceramide dissociates TRAM2 from the ER translocon such that the N-terminus of TM4SF20 can no longer be forced by the first transmembrane domain to function as a signal peptide. Under excess ceramide conditions, TM4SF20 adopts a completely opposite topology and allows the cleavage of CREB3L1 to proceed. We have designated this novel mechanism for transmembrane protein regulation as "alternative translocation."Item Characterization of the Antiviral Effector IFI6(2018-11-26) Richardson, Ryan Blake; Yan, Nan; Schoggins, John W.; Levine, Beth; Pfeiffer, Julie K.The innate immune response is a critical line of host defense against invading pathogens. The production of interferon (IFN) and the subsequent expression of interferon stimulated genes (ISGs) are major contributors to the innate immune response, which establish an antiviral state in the cell. Flaviviruses such as dengue virus, Zika virus, and West Nile virus rely intimately on host pathways for completing a replication cycle, and have developed strategies to overcome the inhibitory effect of the innate immune response. To identify host factors required during an IFN response to flavivirus infection, a genome-wide CRISPR screen was carried out. Two of the top hits from the screen were IFI6, a previously identified ISG long predicted to be antiviral, and BiP, a luminal chaperone in the endoplasmic reticulum (ER). I questioned whether IFI6 was important for the antiviral response to flaviviruses and sought to investigate its role during infection. I confirmed the results from the CRISPR screen and showed that cells lacking IFI6 were insensitive to IFN, suggesting a key role in the innate immune response to flaviviruses. This was complemented by overexpression studies which showed IFI6 is potently inhibitory to flavivirus infection. I further demonstrated that BiP is required for an intact IFN response and importantly mediates expression of IFI6, which it binds in a chaperone-dependent manner. I also showed that IFI6 is localized to the ER and is an integral membrane protein. Importantly, IFI6 acts during the flavivirus life cycle to inhibit replication and formation of replication complexes, which are formed by rearrangement of ER membranes. IFI6 specifically inhibits flaviviruses, since other viruses that replicate at the ER such as hepatitis C virus (HCV) are not affected by IFI6. I hypothesize the key to this specificity lies in the orientation of the replication complexes - HCV complexes extend outwards into the cytoplasm while flaviviruses bud inwards into the lumen. Taken together, these data support a model where IFI6 is sensitive to membrane alterations specifically induced by flaviviruses but not other viruses, which provides the innate immune response with a potent and specific ISG to block viral infection.Item Crosstalk Between Calcium Signaling and Lipid Metabolism at Endoplasmic Reticulum-Plasma Membrane Junctions(2014-04-14) Chang, Chi-Lun; Moe, Orson W.; Roth, Michael G.; Yin, Helen L.; Liou, JenReceptor-induced Ca2+ signaling is the key to many cellular functions, such as secretion, migration, differentiation, and proliferation. The increase in cytosolic Ca2+ signals is dependent on the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane (PM). To enable subsequent signaling activation and maintain cellular homeostasis, it is necessary to replenish the consumed PIP2. However, the molecular mechanisms underlying PM PIP2 replenishment after hydrolysis remain elusive. PIP2 is generated at the PM by sequential phosphorylation of phosphatidylinositol (PI) originating from the endoplasmic reticulum (ER). Delivering PI from the ER to the PM by PI transfer proteins (PITPs) is therefore postulated to support PM PIP2 replenishment. Such transfer is more likely to take place at ER-PM junctions, since the close apposition of the ER and the PM enables PITPs to efficiently interact with two heterologous membranes. To study ER-PM junctions and their roles in PM PIP2 replenishment, I generated a genetically-encoded fluorescent marker to selectively label ER-PM junctions. With this marker, minute ER-PM junctions were easily observed in live cells using multiple imaging techniques. At the resting state, approximately two hundred stable ER-PM junctions were detected at the adhesion surface of a single HeLa cell. Photo-activated localization microscopy (PALM) super-resolution imaging further demonstrate that ER-PM junctions labeled by this marker were remarkably uniform in size and slightly elongated in shape with a long axis of 255.5 nm and a short axis of 157.7 nm. Furthermore, analysis of the distance to nearest neighbor of individual ER-PM junctions show that these junctions are distributed uniformly in the cells. Following the activation of Ca2+ signaling, I observed an enhanced ER-to-PM connection resulting from new junction formation and a decrease in the gap distance of ER-PM junctions. The enhanced ER-to-PM connection depends on cytosolic Ca2+ levels and extended synaptotagmin-like protein 1 (E-Syt1), a C2 domain-containing ER membrane protein. E-Syt1 detects the increase in cytosolic Ca2+ via its C2C domain and translocates from the bulk of ER to ER-PM junctions to enhance ER-to-PM connection. This in turn facilitates the recruitment of Nir2, an ER-associated PITP, to ER-PM junctions to promote PM PIP2 replenishment. In summary, these results indicate a feedback loop for PM PIP2 replenishment via E-Syt1 and Nir2 at ER-PM junctions. Disruption of this feedback mechanism by knockdown of E-Syt1 or Nir2 abolished PM PIP2 replenishment and therefore, impaired receptor-induced Ca2+ signaling. This work reveals the long-sought mechanism of PM PIP2 replenishment following hydrolysis and sheds light on the functional roles of poorly characterized ER-PM junctions. Furthermore, given the fact that PIP2 and Ca2+ are pivotal signaling molecules for many cellular functions, these findings are of significance for providing new mechanistic insights into the signaling crosstalk and may have a broader impact on fields beyond cell signaling, organelle dynamics, and lipid trafficking.Item The Endoplasmic Reticulum Udpase ENTPD5 Promotes Cancer Cell Growth and Survival in the PI3K/PTEN(2011-02-01) Shen, Zhirong; Wang, XiaodongPI3 Kinase and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinase that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. Activation of AKT and downstream PI3 Kinase signaling promotes protein translation, resulting in increased protein flux into ER; this will lead to decreased efficiency of protein folding and accumulation of unfolded proteins in the ER and finally lead to ER stress. How does cancer cell solve this problem of increased folding during rapid growth to avoid ER stress? We discovered that ENTPD5, an endoplasmic reticulum (ER) enzyme, is up-regulated in cell lines and primary human tumor samples with active AKT. AKT upregulates ENTPD5 by relieving transcriptional inhibition by FoxO transcription factors. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN-null cells causes ER stress and loss of receptor tyrosine kinases through ER-associated degradation pathway under stress conditions. Consequently, the growth of PTEN-null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an essential component for PI3K/AKT active cancer cells and a potential drug target for anti-cancer therapy.Item ER-PM Junction Proteins and Their Roles in Regulating Cell Homeostasis and Signaling(2021-05-01T05:00:00.000Z) Quintanilla, Carlo Giovanni; Henne, W. Mike; Schmid, Sandra; Tu, Benjamin; Liou, JenHomeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is a critical step in the phosphoinositide cycle (PI-Cycle). This regulatory feedback mechanism is mediated by the lipid transfer protein (LTP) Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. The work presented in this dissertation, describes the functions of Nir1, a previously unidentified ER-PM junction tether and regulator of Nir2 and PM PIP2 replenishment. Manipulation of Nir1 levels in live cells via overexpression or transient knockdown drives remodeling of ER-PM junction properties. Additionally, Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Importantly, I found that Nir1 localization at ER-PM junctions is a requirement for Nir2 potentiation, highlighting the importance of this subcellular site in regulating the PI-Cycle. The Live-cell and biochemical analysis revealed that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Lastly, I describe a novel localization of Nir proteins near the nucleus and demonstrate the requirement of the minimally characterized domain, DDHD. In summary, the results from these studies identify Nir1 as a novel ER-PM junction tether as well as a positive regulator of the PI-Cycle and of the LTP, Nir2. My observations of Nir proteins near the nucleus implicate a novel subcellular site for phosphoinositide metabolic regulation beyond ER-PM junctions.Item Familial Alzheimer's Disease Mutations in Presenilins Disrupt Endoplasmic Reticulum Calcium Leak(2009-06-19) Nelson, Omar Lloyd; Bezprozvanny, IlyaAlzheimer disease (AD) is the most common form of progressive dementia in adults over the age of 65 years. AD is a fatal brain disease and it currently affects about 27 million people worldwide. It is speculated that the number of people affected by AD will quadruple by 2050. The presence of amyloid beta plaque serves a pathological hallmark for AD, since it was first described by Alois Alzheimer's in 1906. The major risk factors for developing AD are age, mutations in presenilins (PS1 and PS2), mutations in the amyloid precursor protein (APP), cardiovascular diseases, open heart surgery, diabetes, brain injury/head trauma, Apolipoprotein E-e4 (APOE-e4) and the (P86L) mutation in the CALHM1(Calcium homeostasis modulator 1) gene. Multiple missense mutations have been reported in presenilin-1 (PS1), presenilin-2 (PS2) and the amyloid precursor proteins (APP), which are linked to familial AD (FAD). Presenilins are known to function as the catalytic subunit of the (-secretase complex and FAD mutations in presenilins affect APP processing, leading to the accumulation of Aᴲ peptide and amyloid plaque formation in AD brains. In addition to abnormal APP processing, several FAD mutations in presenilins have been linked to abnormal calcium (Ca2+) signaling. Our laboratory recently discovered that presenilin holoproteins function as endoplasmic reticulum (ER) Ca2+ leak channels and that FAD mutations in presenilns affected this function. Our findings potentially provided an explanation for Ca2+ signaling abnormalities resulting from FAD mutations in presenilins. The goal of my thesis project is to establish a connection between presenilins FAD mutations and ER Ca2+ signaling. For these studies we utilized the lipid bilayer reconstitution technique and Ca2+ imaging experiments. In order to establish such a connection I examined the effects of FAD PS1 mutation, FAD PS2 mutation, FAD APP mutation, a mutation in tau and sporadic AD cases on ER Ca2+ leak. In addition, I will map the conductance pore of PS1 using cysteine substitution in transmembrane 6, 7 and 9. These data will help to evaluate the "Ca2+ hypothesis of AD" and will contribute to selecting optimal strategies for treatment of AD.Item Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress(2015-04-14) Woolery, Andrew Ryan; Liu, Qinghua; Orth, Kim; Cobb, Melanie H.; Sternweis, Paul C.The post-translational modification AMPylation is emerging as a significant regulatory mechanism in both prokaryotic and eukaryotic biology. This process involves the covalent addition of an adenosine monophosphate to a protein resulting in a modified protein with altered activity. Proteins capable of catalyzing AMPylation, termed AMPylators, are comparable to kinases in that they both hydrolyze ATP and reversibly transfer a part of this primary metabolite to a hydroxyl side chain of the protein substrate. To date, all AMPylators discovered contain one of two domains: the Fic domain or the adenylyl transferase domain. All currently characterized AMPylators are bacterial in origin and are primarily Type III or Type IV secreted effector proteins, which are injected into a host cell to manipulate host signaling to the microbe's advantage. Examples of these are VopS (Vibrio parahaemolyticus), IbpA (Histophilus somni) and DrrA (Legionella pneumophila). The discovery of SidD, a deAMPylator also from L. pneumophila, shows that this modification is dynamic and could likely have a regulatory role in eukaryotic biology. Supporting this idea is the presence of a single copy of the Fic domain in most metazoans, including humans. The substrates, localization, and function of Fic proteins and other AMPylators in eukaryotic biology are perhaps the largest open questions in this rapidly expanding field. The goal of my dissertation work was to expand the understanding of the effects of AMPylation in eukaryotic signaling. I approached this goal in three ways: by examining the effects of an AMPylator (VopS) with known targets (Rho GTPases) on different aspects of cell signaling, developing screening tools for AMPylation and attempting to elucidate some of the functions of the human AMPylator, FicD, in which the targets are unclear. I found that VopS, in addition to collapsing the host actin cytoskeleton, also inhibits many aspects of host defense signaling including NFB, MAP kinases and the phagocytic NADPH oxidase system. I explored the possibility of other potential substrates of VopS by collaborating on an extensive protein microarray screen for AMPylation, determining that the entire Rho GTPase family is AMPylated. I also discovered that the human AMPylator FicD is induced during the unfolded protein response, is localized to the endoplasmic reticulum and is capable of AMPylating the ER chaperone BiP/GRP78. The progress made in these studies will contribute to understanding the role of this enigmatic modification in mammalian cell signaling.Item Illuminating Organellar and Molecular Organization of the Endomembrane System(2018-07-12) Hsieh, Ting-Sung; Schmid, Sandra; Goodman, Joel M.; Jaqaman, Khuloud; Liou, JenThe endomembrane system consists of virtually all of the structurally and functionally distinct membrane compartments in a eukaryotic cell, except mitochondria and plastids. Different compartments in the endomembrane system communicate with each other through vesicular transport as well as direct membrane-membrane contact. In this work, I first probe spatial organization of membrane contact sites between the endoplasmic reticulum and the plasma membrane in mammalian cells. These membrane contact sites, termed endoplasmic reticulum-plasma membrane junctions, mediate cellular activities ranging from Ca2+ signaling to lipid metabolism. I provide quantitative information on spatial organization of endoplasmic reticulum-plasma membrane junctions and show that it is in part regulated by F-actin. This gives clues about how cellular activities of endoplasmic reticulum-plasma membrane junctions may be regulated because their spatial organization dictates the extent and location of these cellular activities. Then, I examine the phosphoinositide identities of mammalian late endocytic compartments. Phosphoinositides perform the pivotal role as identifiers for different membrane compartments. I show that phosphatidylinositol 3-phosphate is presnt on late endosomes while phosphatidylinositol 4-phosphate is presnt on some endolysosomes and lysosomes. This yields clues about how phosphoinositides regulate membrane sorting and biogenesis of different late endocytic compartments. Last, I show the alteration of phosphoinositide identity of the endoplasmic reticulum by the phosphatidylinositol 3-kinase MavQ secreted by Legionella pneumophila. L. pneumophila exploits the host endoplasmic reticulum membrane to form a Legionella-containing vacuole for intracellular replication. I reveal that MavQ generates phosphatidylinositol 3-phosphate on the endoplasmic reticulum membrane and that MavQ, together with the phosphatidylinositol 3-phosphatase SidP, self-organizes and propagates on the endoplasmic reticulum membrane in a wave-like manner and drives vesicle/tubule generation along the way. This not only provides insight into how L. pneumophila subverts the host cell to establish their own niche but also highlights the importance of concerted kinase/phosphatase integration in generating complex cellular behaviors.Item Metabolic Regulation at Sub-Organelle Length Scales: Inter-Organelle Contacts and Lipid Droplets(2021-09-29) Rogers, Sean W.; Radhakrishnan, Arun; Henne, W. Mike; Liou, Jen; Rosen, Michael K.For cells to properly respond to environmental changes, cellular interiors must be exquisitely organized both spatially and temporally. In particular, metabolism must be spatially coordinated so metabolites are appropriately shunted into either storage or growth. Despite our understanding of how membrane-bound organelles organize metabolic processes, little is known about how metabolic regulation occurs at sub-organelle length scales. At these length scales, physical interactions between the endoplasmic reticulum (ER) and other organelles at ER-membrane-contact-sites (ER-MCSs) are now recognized as sub-organelle hubs for the regulation of metabolic processes. Our work uses the nucleus-vacuole-junction (NVJ) in S. cerevisiae (yeast) as a model ER-MCS to further an understanding about potential general functions of ER-MCSs. We have noted that the NVJ, a physical connection between the nuclear-ER and the vacuole, is a hub for lipid metabolic enzymes and regulators. When yeast are exposed to low glucose conditions, the NVJ recruits several metabolic proteins, including the enzyme Hmg1. Hmg1 catalyzes the conversion of HMG-CoA to mevalonate and is the rate-limiting enzyme in sterol biogenesis. We noted that Hmg1 is less catalytically active when Nvj1, the protein that recruits Hmg1 to the NVJ, is genetically ablated, or when Nvj1 lacks a minimal motif required to recruit Hmg1. Hmg1 NVJ partitioning is accompanied by its assembly into high molecular weight species, which may underlie its increase in enzymatic efficiency. Indeed, artificial tetramerization of Hmg1 overcomes the deficiencies of an Nvj1 knock-out. During Hmg1 partitioning, mevalonate is preferentially shunted into synthesis of sterol-esters (SEs), which are storage lipids found in large cytoplasmic organelles, lipid droplets (LDs). Coordinately, glucose starvation promotes the degradation of triglycerides (TAGs), the other major lipid species contained in LDs. We found that the SE/TAG imbalance in LDs during glucose starvation leads to a phase separation of SEs from a liquid to liquidcrystalline state. Upon SE phase separation, the proteome of LDs is considerably changed. Collectively, our studies of the NVJ have identified a novel function for an ER-MCS and connected it to a lipid metabolic circuit that controls the proteome of LDs.Item Sequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductase(2014-05-28) Morris, Lindsey LaChelle; Goodman, Joel M.; DeBose-Boyd, Russell A.; Lehrman, Mark A.; De Martino, GeorgeAccelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme HMG CoA reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26S proteasomes through poorly defined reactions mediated by the AAA-ATPase VCP/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion, and thus membrane extraction of reductase, was tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.Item Sterol Sensing by Two Luminal Loops in Scap(2014-04-14) Zhang, Yinxin; Thomas, Philip J.; Seemann, Joachim; Roth, Michael G.SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum (ER) membrane protein that controls cholesterol homeostasis by transporting SREBPs from the ER to the Golgi complex. Transport is initiated when COPII proteins bind to Scap and cause the Scap/SREBP complex to enter COPII coated vesicles for transport to the Golgi. In the Golgi complex, two proteases cleave SREBP, thereby releasing its transcriptionally active domain so that it can move to the nucleus and activate transcription of genes involved in cholesterol synthesis and uptake. Scap is not only an escort protein, but also a cholesterol sensor. When cholesterol is abundant in ER membranes, the sterol binds to Scap and triggers a conformational change in the protein that prevents COPII proteins from binding to Scap. The Scap/SREBP complex cannot move to the Golgi and proteolytic cleavage is terminated. This cholesterol feedback inhibition is essential to control cholesterol metabolism in animals. Scap can be divided into two functional regions. The C-terminal cytosolic WD domain interacts with the regulatory domain of SREBPs. The N-terminal membrane attachment domain includes eight transmembrane helices (TM) joined by four small hydrophilic loops and three large loops. One large cytosolic loop (Loop 6) in Scap binds COPII proteins. The other two large loops (Loops 1 and 7) face the ER lumen. Previous studies localized the cholesterol-binding activity to the N-terminal membrane domain of Scap. Studies described in this thesis narrow down the cholesterol binding pocket to the first large luminal loop (Loop 1). Mutational analysis further suggests a direct interaction between luminal Loop 1 and Loop 7 to control Scap transport activity. Scap Loop 1 was purified as a recombinant protein and found to bind [3H]-cholesterol through an in vitro binding assay. The specificity of this binding was determined through competition studies with different unlabeled sterols. Importantly, the binding affinity and specificity of Loop 1 was similar to that of the entire Scap membrane domain. Subsequently, alanine scan mutagenesis was performed on luminal Loop1 and Loop7. Through this approach, two point mutations of Scap (Y234A in Loop 1 and Y640S in Loop 7) were identified that prevent its movement to the Golgi, thus abrogating the processing of SREBPs. Trypsin cleavage assays on the full-length Scap show that Loop 6 of Scap(Y234A) or Scap(Y640S) is always in the configuration that precludes COPII binding, even in sterol-depleted cells. When the Scap TM1-6 segment (containing Loop 1) and the TM7-end segment (containing Loop 7) are expressed in the same cells, the two proteins bind to each other as determined by co-immunoprecipitation. This binding does not occur when Loop 1 contains the Y234A mutation, or Loop 7 contains the Y640S mutation. These data support the model that luminal Loop 1 and luminal Loop 7 must interact in order for Scap movement to occur.Item Structural Studies of Integral Membrane Proteins Involved in GPCR Signaling and Sterol Homeostasis(2018-11-27) Clark, Lindsay D.; Rice, Luke M.; Rosenbaum, Daniel M.; Gardner, Kevin H.; Jiang, YouxingMembrane proteins are crucial molecules for cellular survival, and can take on multiple and diverse roles within the native membrane. In this dissertation, I will detail my efforts to understand and study two different types of membrane proteins. First, I will discuss my research developing and applying a strategy to use NMR spectroscopy to study specific receptors within the large family of G protein-coupled receptors. This strategy enabled the first methyl-TROSY experiments on a wild-type human GPCR, and have significant value for future drug discovery efforts on this important class of membrane proteins. Second, I will discuss my endeavors to understand the important role of the protein Scap, which can both sense and respond to differences in cholesterol levels within the ER membrane. Scap is a central player in the SREBP pathway, which is targeted by multiple classes of pharmaceuticals, including statins. Through efforts described in the second half of this dissertation, I have been able to demonstrate the first biochemical characterization of the full-length mammalian Scap/Insig complex, which has led to the first structural characterization of this important machinery. The long-term goal of both of these projects is aimed at having a more complete understanding of how these important membrane proteins respond to ligands and other environmental changes within their native cell membrane. This information will further our ability to diagnose and treat diseases ranging from insomnia and chronic pain to atherosclerosis and hypercholesterolemia.Item A Study in SCAR20 Neurologic Disorder Reveals Defective Cellular Lipid Homeostasis(2020-11-23) Datta, Sanchari; Goodman, Joel M.; Henne, W. Mike; Schmid, Sandra; DeBose-Boyd, Russell A.Fatty acids (FAs) are important cellular metabolites that are utilized by the cells to perform important functions such as the generation of ATP, membrane biosynthesis, and cell signaling. Dysregulation in FA processing and storage causes toxic FA accumulation which alters membrane compositions and contributes to metabolic and neurological disorders. Excess lipids are stored as lipid droplets (LDs) which sequester toxic FAs and serve as metabolic buffers to maintain lipid and energy homeostasis. LDs emerge from the endoplasmic reticulum (ER) but how their formation is regulated is not completely understood. Recently, we identified sorting nexin family protein Snx14, implicated in cerebellar ataxia disease SCAR20, as a novel factor that enriches at ER-LD contacts following exogenous FA treatment independently of Seipin and promotes FA-induced LD growth. Loss of Snx14 perturbs LD morphology whereas Snx14 overexpression extends ER-LD contacts and promotes LD biogenesis. Proximity-based APEX2 labeling revealed the enrichment of Snx14 at ER-LD contacts during LD biogenesis. Capitalizing on this APEX technology, we also utilize Snx14-APEX2 localization to dissect the protein composition of ER-LD contact sites. We identify proteins involved in fatty acid activation, desaturation, and triacylglycerol synthesis as being enriched at ER-LD contacts, indicating these contact sites serve as lipogenic sub-domains of the ER network. Furthermore, we identify the major delta-9 FA desaturase SCD1 as a key interacting partner of Snx14. Consistent with this, Snx14-deficient cells are hypersensitive to saturated fatty acid (SFA)-mediated lipotoxic cell death that compromises ER integrity. We show that SCD1 is upregulated in SNX14-KO cells, and Snx14-associated SFA hypersensitivity can be rescued by ectopic SCD1 overexpression. The lipid-associated PXA domain of Snx14 and its interaction with SCD1 are required for Snx14-mediated SFA protection function. Snx14 loss mimics SCD1 inhibition and causes accumulation of free FAs and increased membrane lipid saturation. Altogether these mechanistic insights reveal a role for ER-LD contacts as lipogenic ER sub-domains, and Snx14 as an ER-LD tether with a key role in maintaining cellular FA homeostasis through a functional interaction with SCD1, defects of which may underlie the neuropathology of SCAR20.Item Uncovering Reversible AMPylation of BiP Mediated by dFic During ER Homeostasis(2015-01-16) Ham, Hyeilin; Tu, Benjamin; Orth, Kim; Krämer, Helmut; Liu, QinghuaAMPylation is a posttranslational modification involving a covalent attachment of an AMP moiety from ATP to hydroxyl side chains of target substrates. Fic domain which mediates AMPylation is highly conserved across species, including higher eukaryotes, implicating an essential role of this modification in cellular function. Despite the recent discoveries and characterization of a number of bacterial AMPylators and their targets during pathogenesis, the knowledge of AMPylation in eukaryotic system is still elusive. Therefore, the goal of my thesis is to determine the eukaryotic function of AMPylation and identifying the endogenous substrates of this novel modification. In an attempt to understand the physiological function of AMPylation in eukaryotes, we used Drosophila melanogaster as our genetic model organism and created mutant flies lacking functional Drosophila Fic (dFic). We found that the flies without enzymatic function of dFic exhibit blind phenotype due to impaired synaptic transmission. dFic enzymatic activity is required in glial cells for the normal visual neurotransmission. This suggests that a target of dFic may be a component of the visual signaling pathway. dFic was observed in the cell surface of the glial cells particularly enriched in capitate projections. However, dFic is localized to the ER in a number of fly tissues and also in the S2 cells, indicating that there may be another target of dFic in the ER that plays a more general role in the cellular function. In this study, we identified an ER molecular chaperone BiP/GRP78 as a novel substrate for dFic-mediated AMPylation. BiP was predominantly labeled with AMP by dFic in S2 cell lysate. AMPylation of BiP decreases during ER stress but increases upon the reduction of unfolded proteins. Both dFic and BiP are transcriptionally activated upon ER stress induction, implicating a role for dFic in the UPR. We identified a conserved threonine residue, Thr366, as the AMPylation site, which is in close proximity to the ATP binding site of BiP's ATPase domain. Our study presents the first substrate of AMPylation by a eukaryotic protein and proposes a new mode of posttranslational regulation of BiP, which is likely to serve a crucial role in maintaining ER protein homeostasis.Item Variations in Mevalonate Pathway Flux in Human Cells with Familial Hypercholesterolemia(2020-05-01T05:00:00.000Z) Su, Shan; DeBose-Boyd, Russell A.; Liang, Guosheng; Radhakrishnan, ArunBACKGROUND: HMG-CoA reductase (HMGCR) is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One regulator of HMGCR is UBIAD-1, a vitamin K2 biosynthetic enzyme. Individuals with familial hypercholesterolemia (FH) suffer from cholesterol excess due to the inability of cells to take up cholesterol from the environment, leading to a cholesterol depleted cellular state and an increase in cholesterol production. OBJECTIVE: In this study, we examine the effect of sterol and nonsterol isoprenoid depletion via statins followed by mevalonate treatment on the expression of genes and proteins in the mevalonate pathway and localization of UBIAD-1 in human fibroblasts. METHODS: Cells expressing FH mutations and control cells were grown on culture plates or coverslips and fed media containing FCS, or FCS plus compactin and 0.05 mM, 0.2 mM, 1 mM, 3 mM, or 10 mM mevalonate. After overnight feeding, cells were harvested for immunofluorescence visualization, and qRT-PCR and immunoblot analysis of genes and proteins related to cholesterol and nonsterol isoprenoid synthesis. RESULTS: Immunoblot analysis indicates that FH cells generally express higher amounts of sterol biosynthetic enzymes but lower amounts of CoQ10 biosynthetic enzymes than control cells. qRT-PCR showed that genes of the CoQ10 pathway in FH cells are expressed to a significantly less extent than in control cells, and that sterol synthetic genes are relatively unaffected in FH cells but upregulated in control cells fed compactin and mevalonate. Immunofluorescence and quantitation of UBIAD-1 Golgi localization indicate that compactin causes UBIAD-1 to migrate to the ER in both cells, and that FH cells require a greater concentration of mevalonate following the addition of compactin to restore Golgi localization. CONCLUSION: The FH phenotype causes a cellular deficiency of sterols, leading cells to upregulate mechanisms toward sterol synthesis at the expense of CoQ10 synthesis, which results in a relative CoQ10 deficiency in FH cells.