Browsing by Subject "Membrane Proteins"
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Item Autophagy in Zellweger Syndrome Spectrum Disorder and Cancer(2016-05-17) Lee, Ming Yeh; Tu, Benjamin; DeBerardinis, Ralph J.; Hill, Joseph A.; Levine, BethAutophagy is a lysosomal degradation pathway that breaks down unwanted proteins and organelles from the cytoplasm to regenerate cellular building blocks. This process is constitutively active at low basal levels, and can be upregulated by stress stimuli to promote cellular homeostasis. In this work, we investigated two aspects of autophagy regulation and relevance to human diseases. First, we examined how autophagy selectively removes viral components and damaged mitochondria from the cytoplasm through PEX13, a peroxin protein mutated in Zellweger syndrome spectrum (ZSS). Second, we examined the role of autophagy as a potential mechanism contributing to exercise-mediated protection against cancer progression. PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with ZSS disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurodevelopmental, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy), and that disease-associated PEX13 mutants I326T and W313G are defective in mitophagy. The selective mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13-mediated mitophagy may contribute to ZSS pathogenesis. In the second part of this study, we evaluated physiological functions regulated by exercise-induced autophagy, including changes to the metabolome, proteome, and breast cancer progression. A previous study from our laboratory demonstrated that exercise is a potent inducer of autophagy and that autophagy contributes to exercise-mediated metabolic benefits. Therefore, we speculate that autophagy may contribute to exercise-mediated protection against other diseases. Although many epidemiological and laboratory studies have provided strong evidence that physical exercise can decrease cancer development and mortality, the mechanisms are poorly understood. Using the E0771 injectable murine breast cancer, we show that exercise delays cancer progression in wild-type, but not in Bcl-2 AAA mice or Beclin 1 heterozygous knockout mice that are deficient in exercise-induced autophagy. We identified candidate factors and pathways regulated by exercise-induced autophagy, including plasma levels of pyrimidine, branched chain amino acids, LIF, and IL-15, as well as skeletal muscle expression of IDH2 and NDUFA13. Further studies are required to elucidate the metabolomic and proteomic alterations regulated by exercise-induced autophagy and the mechanism by which exercise-induced autophagy protects against tumor progression.Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, John M.Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced an authentic null allele at the Debcl locus. Although gross development and lifespans were unaffected, we found that debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf-1counterpart, dark, but was not required for killing by RPR proteins. Surprisingly, in a model of caspaseindependent cell death, we found that heterologous killing by Murine Bax required debcl to exert its pro-apoptotic activity. DebclKO mutants were also significantly affected for mitochondrial density. Taken together, these findings suggest that evolutionary functions impacting mitochondrial properties represent ancient activities which preceded the evolution of these proteins as central regulators of PCD.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 The CFTR Folding Pathway: Implications for the Identification and Development of CF Therapeutics(2012-07-20) Mendoza, Juan Luis; Thomas, Philip J.The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a member of the ABC transporter superfamily, important for Cl- conductance at the apical cell membrane. Loss-of-function of CFTR leads to Cystic Fibrosis (CF), a fatal genetic disease affecting 70,000 people worldwide. There are hundreds of CF causing mutations with the most common being ΔF508, present in at least one allele in 90% of CF patients. CFTR, comprising of 1480 amino acids, folds into five domains important for forming the channel through the membrane, and the regulation of channel function. F508 is located in Nucleotide Binding Domain 1 (NBD1) and is predicted to be at the interface with Intracellular Loop 4 (ICL4) of Transmembrane Domain 2 (TMD2). Studies of the isolated NBD1 demonstrate that the ΔF508 mutation impacts the folding pathway and stability of the domain. Misfolding of NBD1 contributes to the trafficking defect of the intact protein and subsequent loss-of-function. Conversely, second-site suppressor mutations, which more than compensate for defects of the mutant NBD1 domain, only partially rescue CFTR trafficking, suggesting that the deletion also affects other steps along the folding pathway. The aim of this work was to identify positions in CFTR critical for defining the folding pathway. We used a computational approach and two in vitro folding assays to monitor folding of the isolated NBD1 domain and trafficking of full-length CFTR. These data establish a correlation between the folding of the isolated NBD1 domain and maturation of full-length CFTR. Further, NBD1 second-site suppressor mutations in the ΔF508, F508K (NBD1/ICL4 interface disrupting mutation), and R1070W (ΔF508 NBD1/ICL4 interface stabilizing mutation) backgrounds suggest that ΔF508 CFTR is defective in two steps of CFTR biogenesis: 1) stability and efficiency of folding of the NBD1 domain, and 2) NBD1/ICL4 docking. We demonstrate that efficient rescue of ΔF508 CFTR requires correction the two distinct defects. This work has implications for the discovery and development of CF therapeutics by providing a framework for understanding the observed ceiling in the efficacy of either suppressor mutations or corrector compounds, which likely correct a single defect.Item Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis(2004-05-04) Hays, Lori Beth; Rhodes, Christopher J.; Roth, Michael G.; Cobb, Melanie H.; Uyeda, KosakuInsulin exocytosis is a complex, regulated process involving numerous exocytotic proteins to coordinate the release of insulin. Syncollin has been implicated in zymogen granule exocytosis in acinar cells. It was hypothesized that either syncollin or a ‘syncollin-like’ protein may be expressed in β-cells and influence insulin exocytosis. Adenoviral mediated expression of either long or short forms of syncollin in isolated islets and INS-1 cells showed both forms underwent N-terminal signal peptide cleavage to yield the same 14kD mature protein. Immunofluorescence revealed that adenovirally-expressed syncollin was specifically targeted to the ß-granule lumen. In perifused islets, syncollin expression significantly inhibited first-phase glucose-induced insulin secretion compared to AdV-GFP infected islets. GLP-1 and glyburide potentiation of insulin secretion was inhibited; whereas constitutive secretion and insulin content were normal in syncollin-infected islets indicating syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production or secondary stimulus-coupling signals. Thus, syncollin likely inhibited the distal stages of insulin exocytosis providing the first evidence that an intragranular protein is capable of influencing regulated insulin secretion. Syncollin fluorescent fusion proteins were localized to ß-granules, but did not influence insulin secretion implicating these chimeras as ß-granule specific markers for emerging imaging technology. Real-time confocal microscopy demonstrated syncollin-GFP could be used to examine spatiotemporal dynamics of exocytosis. Furthermore, consecutive infection of syncollin-GFP and syncollin-dsRFP labeled distinct pools of β-granules. Expression of syncollin was not identified in β-cells; however, a 10Kd ‘syncollin-like’ protein was expressed, which when sequenced corresponded to FXYD6. Comparison of syncollin and FXYD6 protein structure revealed several conserved domains, indicating syncollin is likely a pseudo-FXYD family member. FXYD6 was the only FXYD protein endogenously expressed in β-cells, which localized to distinct regions of the plasma membrane. Overexpression of FXYD6-Myc enhanced β-granule transport to distinct regions of the plasma membrane that also expressed FXYD6; however, there was no significant effect on glucose-stimulated insulin secretion in isolated islets. SiRNA-mediated reduction of FXYD6 resulted in no obvious changes in β-granule distribution; however, β-granule movement during glucose stimulation was erratic and misdirected. These data implicate FXYD6 as a molecular beacon on the plasma membrane guiding β-granules to the active site of exocytosis.Item Computer Vision to Characterize Protein Interactions at the Cell Membrane(2018-12-26) Vega, Anthony Raphael; Yu, Hongtao; Jaqaman, Khuloud; Schmid, Sandra; Grishin, Nick V.Protein interactions at the cell membrane provide critical insight into how cells respond to and interact with their environment. Technological advances in light microscopy have allowed an unprecedented perspective into these interactions, however manual analysis of data has become increasingly insufficient to characterize interactions as advances progress. Computer vision tools offer a powerful approach to automate analysis, overcoming limitations of manual analysis to optimize the discovery of novel interactions and their underlying mechanisms. In this thesis, I develop novel computer vision tools to probe the intensity and mobility properties of proteins on the cell membrane, and demonstrate how these can be used to provide insight into membrane protein interactions and organization.Item The Conserved Oligomeric Golgi (COG) Complex Is Required for Normal Import of Fatty Acids in Saccharomyces Cerevisiae(2004-08-19) Ballard, Johnathan L.; Goodman, Joel M.The goal of my work was to elucidate aspects of the mechanism of trafficking of membrane proteins to peroxisomes. The work described in this document centers around one protein from Saccharomyces cerevisiae, Cog7p. Cog7p is part of the conserved oligomeric Golgi (COG) complex. Results describing a basic function of Cog7p were published well after I began studying this protein. Nevertheless, I use the nomenclature outlined in that work. Cog7p functions in intra-Golgi vesicular transport in concert with seven other proteins. This protein complex is found in both yeast and mammals. We found Cog7p in a different context through a screen to identify proteins that function in the trafficking of membrane proteins to peroxisomes. In the screen a portion of Cog7p was found to interact with the membrane peroxisomal targeting sequence, mPTS, of the Candida boidinii peroxisomal membrane protein, Pmp47. I studied peroxisomal biogenesis in a strain of Saccharomyces cerevisiae in which the COG7 gene had been deleted. I showed that Cog7p was not required for peroxisomal biogenesis, but in so doing, established that Cog7p was required for the proper metabolism of fatty acids in a peroxisome-independent manner. I showed that Cog7p was required for the normal import of fatty acids; without Cog7p, yeast cells imported abnormally high amounts of free fatty acid from the environment. My results are consistent with the hypothesis that one or more protein(s) involved in fatty acid import require the COG complex for proper processing. My work ends before such a protein was identified, but I provide leads that if pursued would contribute to understanding the regulation of fatty acid import into yeast cells.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 Elucidation of Molecular Mechanisms Underlying Regulation of Cholesterol Synthesis(2007-05-22) Lee, Peter Chang-whan; DeBose-Boyd, Russell A.Insig-1 and Insig-2, a pair of ER membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to two polytopic ER membrane proteins: SCAP and HMG CoA reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. The successful application of somatic cell genetics in unraveling the SREBP pathway, merits its use in the dissection of mechanisms for Insig-mediated, sterol-accelerated degradation of reductase or ER retention of SCAP. I have designed a genetic screen to isolate mutants of CHO cells that cannot degrade reductase when presented with sterols. CHO cells were mutagenized and selected for growth in cholesterol-free medium containing the SR-12813. SR-12813 blocks cholesterol synthesis by mimicking the action of sterols in accelerating reductase degradation. Using this screen I have isolated the following mutant cell lines. 1) SRD-14 cells, which do not produce Insig-1 mRNA and protein due to a partial deletion of the Insig-1 gene. Sterols fail to promote reductase ubiquitination/degradation and the rate at which sterols suppress SREBP processing is significantly slower in SRD-14 than wild type cells; 2) SRD-15 cells which are deficient in both Insig-1 and Insig-2. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 even upon prolonged treatment; 3) SRD-16, -17, and -18 cells contain a point mutation in one reductase allele. Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs; 4) SRD-19 cells have amplified the number of copies of the gene encoding SCAP, leading to the overproduction of SCAP mRNA and protein. Sterols fail to suppress processing of SREBPs, even though the cells express normal levels of Insig-2. These studies demonstrate 1) absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells; 2) the importance of interactions between Insigs and the membrane domain of reductase in feedback control of a rate-determining step in cholesterol synthesis; 3) the importance of Insig-SCAP ratios in the normal regulation of SREBP processing.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 Intramembrane Proteolysis Mediated by the gamma-Secretase Complex : Nicastrin Functions as a Substrate Receptor(2006-08-11) Shah, Sanjiv; Yu, GangThe proteolytic processing of proteins within the lipid bilayer, and release of their membrane tethered biologically active fragments, fundamentally controls a growing list of cell signaling events. The gamma -secretase, one of a small family of independently evolved proteases, performs this enigmatic hydrolysis of a peptide bond within the membrane. Remarkably atypical, gamma-secretase activity: (1) requires a complex of proteins that include presenilin, nicastrin, Aph1, and Pen-2; (2) catalyzes the intramembrane cleavage of a broad range of substrates, regulating physiology from neurodevelopment to neurodegeneration. The aim of this thesis is to elucidate the mechanism by which the gamma -secretase recognizes its substrates. I provide evidence that nicastrin, in addition to being a critical component of the complex, plays a major function in substrate recognition. The ectodomain of nicastrin binds the new amino terminus that is generated upon the prerequisite 'shedding' of substrates, thereby recruiting substrates into the gamma -secretase complex. The gamma -secretase complex has been traditionally viewed as a hub for signal transduction of substrates such as Notch and APP. The mechanism by which a broad range of substrates may be recognized and subsequently cleaved, as demonstrated in this thesis, supports a mutually inclusive function as a protease that has evolved to simply dispose transmembrane domains thus controlling the repertoire of a class of proteins present in the membrane.Item Logic and Mechanism of an Evolutionarily Conserved Interaction in PDZ Domain(2006-05-15) Sharma, Rohit; Ranganathan, RamaProteins are beautiful materials evolved to channel specific energetic perturbations into particular functions. At the core of virtually every biological process are two features of a protein: the energetic architecture and the mechanisms of energy propagation. Structural, dynamics, and mutagenesis experiments have revealed that anisotropy and cooperativity are common features of the energy propagation in proteins; however, a complete understanding of the patterns and mechanisms of energy propagation remain unclear from these studies. Previous work in our lab developed a methodology, termed the Statistical Coupling Analysis (SCA), to estimate energetic interactions between residues in a protein from their statistical co-variation through evolution. The results of this algorithm revealed a small subset of the residues in a protein have significant energetic interactions and form a connected substructure in proteins and show excellent agreement with mutagenesis data in several systems. Using the same fundamental concepts of the original SCA, we have developed an improved version of SCA. This new algorithm provides, for the first time, a global map of the co-evolutionary interactions between residues in a protein from a multiple sequence alignment. The results of the new SCA are consistent with the original method but produce values for all pairs of positions. We then used the energetic map provided by SCA to understand the physical basis of specificity in the PDZ domain. The co-evolutionary energetic map of the PDZ domain predicts a long range interaction between position 372, a known specificity determinant that directly interacts with ligand, and position 322. Thermodynamic measurements in one PDZ domain reveal that position 322 modulates the specificity-determining interaction between 372 and its ligand contact. Structural studies show that flexibility at 322 is tuned to make conformational change on one side of the binding pocket sensitive to interactions at the distant specificity-determining contact. This designed mechanical coupling allows the domain to have AND gate-like behavior in screening for specific binding interactions. Understanding the logic and mechanism of a co-evolved interaction gives confidence in the ability of SCA to identify the functionally critical interactions in a protein, even when not structurally obvious. Given the functional and structural relevance of SCA predictions, we next addressed the topology of the energetic map in proteins. Analysis of several structurally and functionally diverse proteins revealed several common striking features in their energetic maps. First, the highly co-evolved positions in a protein show a high degree of mutual co-evolution so that, together, they form a nearly completely co-evolved sub-cluster. Secondly, the pattern of energetic interactions in proteins is highly heterogeneous, and fit a power-law distribution where most residues have very few co-evolutionary links with other residues and a few residues have many co-evolutionary links. The data is very consistent with extensive mutagenesis studies in several systems. Together, these experiments begin to demonstrate that the contiguous networks identified by SCA reflect structural regions capable of cooperatively channeling energy to produce functionality.Item Measuring Activation of the Cytosolic DNA Sensing Pathway(2019-04-15) Varnado, Nicole L.; Tu, Benjamin; Beutler, Bruce; Cobb, Melanie H.; Chen, Zhijian J.In mammalian cells, DNA is normally sequestered within the confines of the nucleus or mitochondria. Entrance of DNA into the cytosol, whether foreign or self in origin, acts as a danger signal that triggers a host innate immune response. Cytosolically localized DNA is sensed by cyclic GMP-AMP synthase (cGAS), which synthesizes a novel second messenger known as cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP, in turn, binds to and activates the ER resident adaptor Stimulator of Interferon Genes (STING), which triggers downstream signaling that culminates in the production of type-I interferons and other immune modulatory molecules. The pathway underlies the recognition of pathogenic DNA necessary to quell microbial infections, as well as the aberrant detection of self-DNA responsible for inducing certain autoimmune diseases. Such appreciation for the involvement of cGAS-cGAMP-STING signaling in numerous clinical phenotypes necessitates development of tools that can outline the extent of its contribution to various diseases. Additionally, numerous questions remain regarding the regulation of cGAS-cGAMP signaling. As 2'3'-cGAMP production is a hallmark of the pathway's activation, we sought to develop a robust method to monitor its formation in vivo, and quantify its levels in a wide variety of settings. Herein we present the development of an antibody of high sensitivity and specificity for this small molecule second messenger, capable of recognizing and quantifying 2'3'-cGAMP production in vivo. We show it can be adapted for use in a variety of techniques, to track and measure levels of 2'3'-cGAMP quantitatively, to visualize 2'3'-cGAMP produced in cells, and to quickly identify cGAMP-positive cell populations within live samples. We show this antibody to be an invaluable tool to elucidate outstanding questions in the field, and demonstrate its potential to detect patients with aberrant activation of the cGAS-STING pathway. We foresee a future in which the 2'3'-cGAMP antibody is used to quantify activation of the cGAS pathway in a variety of clinical and research settings.Item The Mechanism and Function of Autophagy Induction by Cytosolic DNA(2018-07-11) Gui, Xiang; Olson, Eric N.; Hooper, Lora V.; Wang, Zhigao; Chen, Zhijian J.Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects pathogen infections or tissue damage by binding to microbial or self-DNA in the cytoplasm. Upon binding to DNA, cGAS produces cGAMP that binds and activates the adaptor protein stimulator of interferon genes (STING), which activates the kinases IKK and TBK1 to induce interferons and other inflammatory cytokines. Here, we report that STING also activates autophagy and induces cell death through a mechanism independent of TBK1 and IRF3 activation, which canonically triggers innate immunity signaling. Upon binding to cGAMP, STING translocates to the ER-Golgi intermediate compartments (ERGIC) and the Golgi in a process that depends on the COP-II complex and ARF GTPases. The STING-containing ERGIC serves as a membrane source for LC3 lipidation, a key step in autophagosome biogenesis. Interestingly, STING lacking its C tail for interferon signaling is still capable of membrane trafficking and autophagy induction. Through endosomes or autophagosomes, STING is further degraded in the lysosome to shut down its activation. Interestingly, we determined that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Furthermore, sea anemone STING induces autophagy but not interferons in response to stimulation by cGAMP, suggesting that induction of autophagy is a primordial function of the cGAS-STING pathway.Item The Mechanism of cGAS-STING Signaling in Antiviral Immunity(2021-05-01T05:00:00.000Z) Li, Minghao; Fu, Yang-Xin; Beutler, Bruce; Chen, Zhijian J.; Hooper, Lora V.Unlike inflammatory cell death pathways, apoptosis is a highly regulated process that leads to cell death without the secretion of pro-inflammatory cytokines. The intrinsic apoptosis pathway (IAP) is triggered upon sensing cellular stress, activating pro-death B cell lymphoma 2-associated X (BAX) and B cell lymphoma 2 homologous antagonist killer (BAK) proteins. BAX and BAK induce mitochondrial outer membrane permeabilization (MOMP), spilling cytochrome C (cyt C) into the cytoplasm where it forms the apotosome complex with the apoptotic protease activating factor 1 (Apaf-1). Apaf-1 subsequently induces a cysteine-aspartic protease (caspase) activation cascade culminating in activation of caspases-3/7. Nevertheless, the purpose of the caspase activation cascade remained an interesting conundrum as BAX and BAK, not Apaf-1 and caspases, are necessary for induction of apoptosis. Recent publications seem to indicate that the main function of caspases after activation of apoptosis is to prevent the induction of type I interferons by cyclic GMP-AMP synthase (cGAS) or stimulator of interferon genes (STING) to maintain an immunologically quiescent cell death. I utilized the drug ABT-737 to inhibit the B-cell lymphoma (BCL) family of proteins to prevent the activation of BAK and BAX, leading to MOMP. MOMP causes leakage of cyt C and mitochondrial DNA (mtDNA), activating Apaf-1 and cGAS, respectively. cGAS catalyzes the creation of the cyclic dinucleotide cyclic GMP-AMP (cGAMP) to activate STING which activates downstream targets such as TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) to induce the expression of interferonβ (IFNβ) and interferon-stimulated genes (ISGs) such as C-X-C motif chemokine ligand 10 (CXCL10). However, the mechanism behind caspase-dependent inhibition of the cGAS-STING pathway is currently unknown. My central hypothesis is that caspases-3/7 activate a downstream regulator that inhibits cGAS-mediated cGAMP production. These findings will further our understanding of the regulatory mechanics behind immune responses. As STING is necessary for the production of IFNs downstream of cGAS, it plays an important role in activating the innate immune response during infections. Specifically, mice lacking cGAS or STING are highly susceptible to acute herpes simplex encephalitis (HSE). STING-induced type I IFNs and immune-priming of other cell types were suggested to be critical for protecting mice from HSE. Recent work from our lab has identified an additional primordial downstream pathway of STING: autophagy; in addition, these results indicate that the interferon- and autophagy-inducing function of STING can be uncoupled. To better understand how STING-induced autophagy is controlled, I investigated the necessity of certain canonical autophagy-related genes. To also understand the exact mechanisms downstream of STING activation required for antiviral immunity, I utilized several different STING mutant mice generated in our lab and assessed their response to HSV-1 infection. My results indicate that a novel interferon-independent function of STING was essential to protect against infection and improve the overall survival rates of infected mice; however, STING-induced autophagy was not sufficient to protect against viral challenge.Item Mechanisms of Genome Buffering and Cell Fate Coordination in Adult Tissue Homeostasis(2016-07-26) Tuladhar, Rubina; Amatruda, James F.; Scherer, Philipp; DeBerardinis, Ralph J.; Lum, LawrenceSelf-renewal competency of adult stem cells is essential for tissue homeostasis. The corruption of genes essential for genome preservation or for niche-stem cell interactions frequently results in loss of stem cell viability and disease. The two components of my thesis focus on understanding adult stem cell preservation - the integration of metabolism and intercellular communication mediated by the Wnt family of secreted signaling molecules, and epigenetic mechanisms that buffer the proteome against insertion/deletion (INDEL)-type genetic mutations. Wnt-mediated signaling is essential for embryogenesis and the maintenance of adult tissues. Lipidation of Wnt proteins by the acyltransferase Porcupine (Porcn) is crucial for secretory pathway exiting. Using chemically based approaches, I have demonstrated that Porcn active site features conserved across animals enforce ω-7 cis fatty acylation of Wnt proteins. Deviant acylation of a Wnt protein using an exogenously supplied trans fatty acid cripples its ability to traverse the secretory pathway due to a previously unappreciated stereoselectivity of the Wnt chaperone Wntless (WLS) for fatty acids. My findings provide a mechanistic account of chemical specificity observed in Porcn inhibitors, and delineate a universal mechanism for integrating communal cell fate decision-making with metabolic fitness. As part of my efforts to generate isogenic cells for the expression of LKB1, a tumor suppressor that regulates Wnt protein production, I encountered the emergence of foreign LKB1 proteins subsequent to the introduction of INDELs by the DNA editing enzyme CRISPR-Cas9. I demonstrate that these novel proteins are the products of: a) the installation of internal ribosomal entry sites (IRES), b) the induction of exon skipping due to compromised exon splicing enhancers (ESEs), and c) the conversion of pseudo-mRNAs to protein-coding mRNAs due to the unwanted elimination of premature termination codons. I propose that these molecular events serve as compensatory mechanisms employed by cells to restore proteome integrity in the face of INDEL-type challenges to the genome posed by pathogens and environmental mutagens. Taken together, these two projects will: a) delineate intervention strategies premised upon the attack of an universally conserved point of intersection between metabolism and cell-to-cell communication, b) facilitate the personalization of medicine, and c) accelerate tissue engineering initiatives.Item Mechanistic Dissection of Insig-1, a Master Regulator of Cholesterol Homeostasis(2006-05-15) Gong, Yi; Brown, Michael S.Insigs are polytopic membrane proteins of the endoplasmic reticulum (ER) that regulate lipid synthesis by controlling the sterol-mediated vesicular transportation of sterol regulatory element binding proteins (SREBPs). SREBPs are ER bound transcription factors that form complexes with Scap. In sterol-depleted cells, Scap escorts SREBPs from the ER to the Golgi apparatus, where SREBPs are proteolytically cleaved to liberate the nuclear fragments that activate genes for cholesterol synthesis and uptake. When sterols overaccumulate in cells, the Scap/SREBP complex is retained in the ER by the anchor proteins called Insigs. In this thesis I describe the formation of a complex between Insig-1 and Scap in a sterol regulated fashion which facilitates the ER retention of Scap. To understand the molecular basis of the interactions between Insig-1 and Scap, I use a site-directed mutagenesis approach to select residues in Insig-1 that are essential for Insig-1/Scap complex formation. This study reveals a functional role for the amino acid Asp-205, which is located at the beginning of the fourth loop of Insig-1. Mutation of this aspartic acid to alanine produces an inactive Insig-1 that no longer binds to Scap, and leads to sterol-resistant processing of SREBPs. Mammalian cells express two Insig proteins differ in their mode of control. Insig-1, but not Insig-2, is an SREBP target gene. Also, Insig-1 protein is degraded more rapidly than Insig-2. Thus, Insig-1 is the focus of the study. I further demonstrate that degradation of Insig-1 is regulated by sterols. When ER cholesterol content is low, Insig-1 is ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Sterol-induced binding of Insig-1 to Scap prevents Insig-1 ubiquitination and degradation. The dynamic change in Insig-1 protein stability, together with its transcriptional control by nuclear SREBPs, creates a new model for the convergent inhibition of SREBP processing and cholesterol supply in animal cells. Taken together, these studies established Insig-1 as the master regulator in the cholesterol homeostasis.Item Neurologin Function in Excitatory and Inhibitory Synapses(2008-09-19) Zang, Tong; Südhof, Thomas C.Neuroligins (NLs) are postsynaptic cell adhesion molecules which by binding to presynaptic neurexins (NRXs) are thought to mediate synapse formation and function. Both NLs and NRXs are discussed in the genetic correlation to Autism. Over-expression of NLs could induce the formation of synaptic contacts with axons in non-neuronal cells and increase the synaptic density and response in cultured neurons, through binding and recruiting NRXs; however, little is known about NL signaling though NRXs or inside the cell. First, we hypothesized that NLs signal through their cytoplasmic region. Over-expression of NL1 with cytoplasmic tail truncation abolished the increase of synaptic density by NL1 full length. By yeast two hybrid screening using NL2 cytoplasmic region, we identified potential interaction partners, of which Necab2 and NRP/B (also named as ectodermal cortex 1, EC1) are two promising candidates and the interactions were confirmed. NL1 or NL2 c-tail truncations partially abolished the change in miniature IPSC, but not the evoked responses. NL c-tail binding partners?ver-expression does not show any change in evoked responses. It suggested that NL cytoplasmic region is important for some neuronal changes but does not contribute to the major phenotype of NLs. And we investigated the contribution of NL-NRX binding by using NL extracellular NRX binding mutants. The mutants abolished the change of the evoked and miniature inhibitory responses from the NL2 wild type, which suggested the inhibitory responses triggered by NL2 go through NRXs. And the slight change of the paired pulse ratio suggested the change of presynaptic calcium through binding. The study suggested that NL2 facilitate the inhibitory synaptic transmission through extracellular region via neurexin binding, possibly by the increase in presynaptic calcium. We also found Brain-specific Angiogenesis Inhibitors (BAIs), a family of G-protein coupled receptors (GPCRs), will bind to NLs extracellularly and may serve as signaling modules binding to NLs. Over-expression of BAIs do not change evoked IPSCs, but Bai1 decreased evoked EPSCs and increased the burst duration in the spontaneous responses, possibly because of some secondary responses. Therefore, we found NL-NRX though NL extracellular region is important for NL2 function in synaptic transmission, and BAIs may be potential signaling molecules of NLsItem Phase Transitions of Multivalent Adaptor Proteins(2015-05-27) Banjade, Sudeep; Rice, Luke M.; Rosen, Michael K.; Liou, Jen; Ross, Elliott M.Eukaryotic cells efficiently organize their activities to achieve their functional capabilities. This organization of biochemical reactions is a direct result of the cells' ability to compartmentalize their molecules. For example, within a eukaryotic cell, compartments like the nucleus, the endoplasmic reticulum and the vacuoles exist, which are relatively well known for their specific functions. These aforementioned compartments are surrounded by membranes. However, for the past hundred years, we have also known about assemblies of biomolecules that are not bound by membranes. After the initial discovery of nuages, other structures such as Cajal bodies, the nucleolus, promyelocytic leukemia (PML) bodies, paraspeckles, etc., were also described as membraneless organelles. Furthermore, membranes themselves are self-assembled entities of lipids, proteins and carbohydrates. Additionally, within and on surface of membranes, molecules cluster into signaling compartments in many different biological pathways. Interactions between individual biomolecules have been studied comprehensively in biology. One of our goals as biophysicists is to attempt to propose physical properties that allow these interactions at the subnanometer scale to give rise to formation of cellular structures, the compartments that are listed above. This thesis proposes a hypothesis based on polymerization of multivalent proteins that causes these complexes to phase separate in solution. The behavior of multivalent proteins and their ligands to phase separate may be a general property that allows cells to regulate their activities in certain localized compartments. To study this larger goal, I used a specific example of proteins involved in creating the slit-diaphragm, which is the filtration barrier of our kidneys. Nephrin, an integral membrane protein at the slit-diaphragm, interacts with its partners Nck and N-WASP in a multivalent fashion. I show here that these interactions create large assemblies that phase separate into liquid droplets, both in solution and on membranes. I also find that the creation of these assemblies affects the downstream biochemical activity of N-WASP toward the Arp2/3 complex and actin. The widespread existence of multivalent molecules suggests that these findings may have broad corollaries in different biological systems.Item Regulation of Hepatic Cholesterol Homeostasis Through Accelerated Degradation of HMG CoA Reductase(2017-04-06) Hwang, Seonghwan; Liang, Guosheng; DeBose-Boyd, Russell A.; Bruick, Richard K.; Scherer, PhilippCholesterol biosynthesis is rigorously controlled by negative feedback regulation. This reaction occurs, in part, through sterol-accelerated degradation of HMG CoA reductase (HMGCR), which catalyzes the rate-limiting step in cholesterol biosynthesis. The molecular mechanisms for the degradation of HMGCR have been actively investigated; however, the physiological relevance of the degradative regulation in animals is unclear. The current study investigates the role of sterol-accelerated degradation of HMGCR in overall regulation of HMGCR protein and cholesterol homeostasis in the liver. This was achieved by utilizing two mouse models: (1) liver-specific transgenic mice expressing the membrane domain of HMGCR, which is necessary and sufficient for sterol-regulated degradation of HMGCR in cultured cells and (2) knock-in mice expressing mutant HMGCR that is resistant to sterol-induced ubiquitination. These models were subjected to various feeding regimens known to modulate Insig and Scap, key players in feedback regulation of HMGCR. Cholesterol replenishment accelerates degradation of HMGCR in the liver of transgenic animals, whereas deprivation of sterols by lovastatin administration suppresses degradation of HMGCR. Ubiquitination-resistant HMGCR accumulated in the liver and resulted in the elevation of hepatic cholesterol, indicating degradation plays a significant role in the in vivo regulation of the enzyme and cholesterol homeostasis. This study further explored the physiological settings other than changing cholesterol status that may modulate the degradation of HMGCR in the two mouse models. As cholesterol synthesis is an oxygen-consumptive process, I determined the link between oxygen sensing and feedback control of cholesterol synthesis. In cultured human fibroblasts, stabilization of oxygen-sensitive transcription factor, hypoxia-inducible factor-1α (HIF-1α) directly activates transcription of INSIG-2 gene. Insig-2 inhibits cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent degradation of HMGCR. Hepatic levels of Insig-2 mRNA are enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in liver stimulates HMGCR degradation through a reaction that requires the protein's prior ubiquitination and the presence of Insig-2. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events in the cellular adaptation to hypoxic stress. Overall, the current study provides evidence supporting the physiological significance of the accelerated degradation of HMGCR in cholesterol homeostasis.