Browsing by Subject "Membrane Proteins"
Now showing 1 - 17 of 17
Results Per Page
Sort Options
Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, JohnBcl-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., M.D.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. NPC1Õs sterol binding domain was localized to itÕs 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. [Keywords: Niemann-Pick C Disease; cholesterol trafficking; cholesterol binding; oxysterols; lysosomes; Niemann-Pick C1 protein; Niemann-Pick C2 protein]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. [Keywords: protein folding disease, membrane protein folding, cystic fibrosis, bioinformatics, statistical coupling, X-ray structure, correction mechanism, mechanism of drug action]Item Characterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis(2004-05-04) Hays, Lori Beth; Roth, MichaelInsulin 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 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 Deciphering the Mechanism of E. coli tat Protein Transport: Kinetic Substeps and Cargo Properties(2012-12-03) Whitaker, Neal William 1982-The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. The E. coli Tat machinery minimally consists of three integral membrane proteins: TatA, TatB and TatC. A popular model of Tat translocation is that cargo first interacts with a substrate binding complex composed of TatB and TatC and then is transported across the inner membrane through a channel comprised primarily of TatA. The most common method for observing the kinetics of Tat transport, a protease protection assay, lacks the ability to distinguish between individual transport sub-steps and is limited by the inability to observe translocation in real-time. Therefore, a real-time FRET based assay was developed to observe interactions between the cargo protein pre-SufI, and its initial binding site, the TatBC complex. The cargo was found to first associate with the TatBC complex, and then, in the presence of a membrane potential (?psi), migrate away from the initial binding site after a 20-45 second delay. Since cargo migration away from the TatBC complex was not directly promoted by the presence of a ?psi, the delay likely represents some preparatory step that results in a transport competent translocon. In addition, the Tat system has long been identified as a potential biotechnological tool for protein production. However, much is still unknown about which cargos are suitable for transport by the Tat system. To probe the Tat system?s ability to transport substrates of different sizes and shapes, 18 different cargos were generated using the natural Tat substrate pre-SufI as a base. Transport efficiencies for these cargos indicate that not only is the Tat machinery?s ability to transport substrates determined by the protein?s molecular weight, as well as by its dimensions. In total, these results suggest a dynamic translocon that undergoes functionally significant, ?psi-dependent changes during translocation. Moreover, not every protein cargo can be directed through the Tat translocon by a Tat signal peptide, and this selectivity is not only related to the overall size of the protein, but also dependent on shape.Item Elucidation of Molecular Mechanisms Underlying Regulation Of Cholesterol Synthesis(2007-05-22) Lee, Peter Chang-whan; DeBoseBoyd, 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 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 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; Sudhof, 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 The Role of Insig-Mediated Cholesterol Homeostasis in Mouse Hair Development(2012-08-15) Farooqi, Midhat Saleem; Brown, MichaelInsig-1 and Insig-2, two very homologous proteins, are indispensable for feedback inhibition of cholesterol biosynthesis. Mice null for both Insigs drastically overproduce cholesterol and its precursor sterol intermediates and exhibit many developmental abnormalities such as cleft palate. In this work, we generate and characterize a line of mice lacking both Insigs in the hair and skin. These epidermal-specific, Insig-double knockout mice have many skin abnormalities, but their most striking defect is a complete lack of body hair. Epidermal-specific, Insig-double knockout mice also have a significant buildup of cholesterol precursors in skin, as they are unable to check endogenous cholesterol production. However, topical treatment of these mutant mice with simvastatin, an inhibitor of cholesterol biosynthesis, can reduce these sterol intermediates and completely correct the skin defects and alopecia. Further studies of epidermal-specific, Insig-double knockout mice showed that they had a dramatic decrease in the expression of many keratin-associated proteins relative to their control littermates. In the case of certain keratin-associated proteins, this loss of mRNA was especially severe; keratin-associated protein 28-13 expression, for example, was reduced by more than twenty-fold. Electron microscopy revealed that the hair shafts of mutant mice had grossly abnormal cuticles, and topical treatment of mutant mice with simvastatin rescued keratin-associated protein expression. We conclude that epidermal-specific, Insig-double knockout mice have a hair eruption defect due to improper formation of the hair shaft cuticle likely caused by a lack of keratin-associated protein expression. These findings are relevant to the multiple skin and hair abnormalities seen in human diseases where sterol precursors accumulate due to inborn errors in cholesterol biosynthesis. [Keywords: insig; hair development; hair cuticle; cholesterol; sterol intermediates; keratin-associated proteins; Krtaps; simvastatin; statin]Item Role of Insig-Mediated Cholesterol Homeostasis in the Development of the Palate and Hair Follicles in Mice(2012-08-15) Goldstein, Joseph L.; Brown, Michael S.Proper cholesterol homeostasis is crucial in mammalian development. Currently, there are 8 known human malformation syndromes due to genetic defects in cholesterol biosynthesis. Patients with these syndromes present with a constellation of developmental defects, ranging from skeletal and craniofacial dysmorphologies to skin and cardiovascular anomalies. In each of these diseases, there is a block at a specific enzymatic step leading to a deficiency in cholesterol and a concomitant buildup in sterol intermediates proximal to the block; however, it is unclear whether cholesterol deficiency or sterol intermediate accumulation causes the observed abnormalities. In this work, we generated mice lacking Insig-1 and Insig-2. These proteins regulate cholesterol biosynthesis by both inhibiting the proteolytic activation of SREBPs, transcription factors for cholesterol biosynthetic genes, and enhancing the degradation of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. We first created Insig-double-knockout (Insig-DKO) mice homozygous for null mutations in Insig-1 and Insig-2. Insig-DKO mice exhibited defects in midline facial fusion, ranging from cleft palate to complete cleft face, and overproduced sterols, creating a significant buildup of cholesterol and sterol intermediates. When Insig-DKO mice were treated with lovastatin, an HMG-CoA reductase inhibitor, sterol intermediate levels were significantly reduced and the craniofacial defects were ameliorated. Next, we generated mice lacking Insigs in the epidermis (Epi-Insig-DKO). All Epi-Insig-DKO mice failed to grow body hair, had decreased body weights, and died 6 weeks after birth. Early hair follicle development was normal while later development was disrupted and led to hair cycle arrest in these mice. Skin from these mutant mice showed a significant buildup of both cholesterol and sterol intermediates. Topical treatment of Epi-Insig-DKO mice with the HMG-CoA reductase inhibitor simvastatin during the first two postnatal weeks lowered sterol intermediate levels in the skin and completely corrected the hairless and low body weight phenotypes. We conclude that the accumulation of sterol intermediates, not a deficiency of cholesterol, interferes with both the fusion of facial structures and the proper development of hair follicles in mice. Our findings have implications for the pathogenesis of the palate and skin/hair follicle defects observed in human malformation syndromes due to aberrant cholesterol biosynthesis.Item SCAP, Insig, and Cholesterol Interactions in Mammalian Cells(2007-05-22) Feramisco, Jamison Derek; Brown, Michael S.Cholesterol synthesis in mammalian cells is highly regulated by an end-product feedback mechanism. The transcription of genes necessary for both fatty acid and cholesterol production are controlled by sterol regulatory element binding proteins (SREBPs). The critical regulatory step is the proteolytic release of SREBPs from their inactive membrane bound form. Soon after translation, SREBPs bind SREBP cleavage activating protein (SCAP), a polytopic membrane protein of the endoplasmic reticulum (ER). In sterol depleted situations, SCAP escorts SREBPs to the Golgi, where SREBPs are cleaved and can move freely to the nucleus and activate the numerous enzymes of cellular lipid homeostasis. When cellular sterol levels rise, the SCAP/SREBP complex binds to an ER resident protein named Insig. Upon binding to Insig, the movement of the SCAP/SREBP complex to the Golgi is inhibited, thus halting cholesterol and fatty acid synthesis. The mechanism by which the cell senses sterol levels has been long unknown. Radhakrishnan et al. and Adams et al. demonstrated that SCAP itself binds cholesterol and thus may act directly to sense cellular sterol levels and mediate the end-product feedback control of SREBPs. The goal of this thesis is to elucidate the molecular details of the interactions between SCAP, Insig and cholesterol. My thesis experimentally details the membrane topology of human Insig-1 and shows that it is a polytopic integral membrane protein of the ER with six transmembrane spanning segments. In addition, the amino and carboxy-termini of Insig are both facing the cytosol. Furthermore, crucial residues of Insig that are important for SCAP interaction are identified. My thesis has also defined distinct amino acids of SCAP that are essential for its role as a protein that binds Insig and as a protein that has the ability to bind cholesterol. An aspartic acid in the middle of transmembrane six is necessary for sterol regulated binding to Inisg, while residues in transmembrane segments one and three of SCAP are crucial for cholesterol binding both in vivo and in vitro.Item Supported Lipid Bilayer Electrophoresis: A New Paradigm in Membrane Biophysics and Separations(2012-11-28) Pace, Hudson 1982-The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind?s understanding of the cellular membrane.Item Synaptic Cell Adhesion And Functional Architecture Of CNS Synapses(2007-08-08) Atasoy, Deniz; Kavalali, Ege T.Synapses are specialized intercellular junctions through which neurons communicate. The two sides of a synapse are held together with adhesion molecules. We investigated the role of synaptic adhesion molecules neuroligins, neurexins, SynCAM and dystroglycan using overexpression and knock-out mice analysis approaches. We showed that neuroligins mediate validation of synapses in an activity dependent manner and different isoforms of neuroligins mediate different types of synapse validation. We also showed that binding partners of neuroligins, i.e. neurexins, have a cell autonomous effect on inhibitory synapses, independent of neuroligins. Analysis of dystroglycans failed to reveal a significant phenotype in dissociated cultures, whereas SynCAM had a robust synapse inducing role in developing networks. Furthermore we have shown that this effect of SynCAM is mediated through its cytoplasmic interactions. We further investigated cytoplasmic downstream effectors of neurexins and SynCAM by analyzing CASK and Mint proteins using knock-out approaches. We have shown that both CASK and Mints are essential for survival and synaptic function. Our results indicate that synaptic cell adhesion molecules are not merely passive structural elements but actively participate in information transfer and signaling between neurons by interacting with each other and with their intracellular effectors. We have also investigated homeostatic properties of vesicle recycling and we have demonstrated that activity levels of a neuronal network determines the pathways through which synapses replenish the neurotransmitter vesicles during high frequency stimulation. Finally, we explored the relationship between evoked and spontaneous vesicle fusion and their postsynaptic targets using NMDA receptors. We uncovered previously unexpected segregation of receptor pools that respond to spontaneous or evoked vesicle fusion events. Our experiments also revealed that synaptic cleft is not a mere empty space but rather a complex structure filled with various elements can effect diffusion of neurotransmitter and hence information transfer between neurons.Item Transmembrane Protein Folding: Effects of Disease-Causing Mutations on CFTR Folding and Assembly(2006-05-16) Thibodeau, Patrick Harlan; Thomas, Philip J.The biosyntheses of multi-domain membrane proteins are complex processes which involve the translation, folding, and assembly of domains to reach the native state. The nascent chain of a membrane protein must interact with multiple solvent environments, ribosome and chaperone components, and processing and trafficking machinery, and each of these steps are at least partially determined by the protein sequence and structure. Alterations to protein sequences often perturb these processes by impacting any of a number of structural states of the protein, and while some mutations impact the native state structures of proteins directly, others impact the folding process and have little direct effect on the native state structure. A growing number of mutations have been shown to impact these folding processes in the cystic fibrosis transmembrane conductance regulator (CFTR), a multi-domain transmembrane protein associated with cystic fibrosis. Two such mutations are detailed in this work: F508del and P205S. The most common CF-causing mutation, F508del, is the deletion of a single phenylalanine residue in a cytosolic domain of CFTR and results in a protein which fails to fold at physiological temperature, is retained in the ER and is degraded by the proteasome. The resulting loss of protein is the underlying basis for cystic fibrosis. The loss of the backbone at this position induces the misfolding of the domain, while changes in sidechain character impact subsequent domain-domain assembly. The rescue of full-length F508del CFTR by second-site suppressors correlates with the rescue of the folding of the soluble domain, further suggesting the direct role of Phe508 in domain folding. The mutation of equivalent residues in homologous proteins results in similar phenotypes, suggesting an evolutionary conservation of function for this position. The Pro205 residue, in the first transmembrane domain, has been shown to facilitate proper folding by disfavoring alternate, non-native protein conformations. A computational study of proline residues in transmembrane helices suggests that this mechanism is also conserved evolutionarily. With these data, a hierarchical model for CFTR folding is presented and mechanisms by which these mutations specifically impact the stepwise folding and assembly of CFTR are suggested.