Browsing by Subject "Receptors, LDL"
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Item Analysis of the Function of Megf7 During Development(2007-08-08) Johnson, Eric Boyd; Herz, JoachimLrp4/Megf7, a member of the low-density lipoprotein receptor gene family, is a major regulator of tissue patterning during embryonic development. Prior to this work, the function Megf7 had largely been unknown. Eight distinct mutations in Megf7 were introduced into mice in order to facilitate a functional analysis of Megf7. The Megf7EC Stop mutant, which has a premature stop codon inserted upstream of the transmembrane domain, had defects in limb, tooth, and mammary gland development. Subsequent analysis showed that the defects in limb development were caused by the expansion of the limb bud structure called the apical ectodermal ridge. Biochemical analysis suggests that Megf7 acts as an inhibitor of the Wnt signaling pathway both in vivo and in vitro. The analysis of the tooth defect of the Megf7EC Stop mutant shows that there is an increase in BMP activity, one of the major cellular signals involved in development. This increase in BMP activity leads to a loss of patterning during tooth development. The complete Megf7 null allele that was generated, in addition to having limbs defects, also failed to form kidneys and was paralyzed at birth. The kidney agenesis is the result of a failure of the ureteric bud to grow and branch during the initial stages of kidney development. The phenotype of the Megf7EC Stop and Megf7KO mutants prompted us to look for natural mutants in other animals. We found that Mulefoot Disease, a form of syndactyly in cows, is caused by a single base change at the exon/intron border of exon 37 of bovine Megf7. in vivo and in vitro data suggests that this mutation leads to altered splicing of the gene and premature truncation of the translated gene product. The other six mutant mouse alleles that were generated had specific mutations introduced into the cytoplasmic domain of Megf7. The limb phenotypes of these mice suggest that Megf7 may serve as an endocytic receptor. This work establishes Megf7 as a major regulator of patterning during development and is involved in a natural form of limb dysgenesis. This work will serve as the groundwork for future analysis of Megf7.Item Animating the LDL Receptor Pathway(2007-05-23) Van Exel, Kimberly; Calver, Lewis E.The main goal of this thesis was to animate the LDL receptor pathway for use during lectures and presentations. These animations show how cholesterol-carrying LDL binds with the LDL receptor to be endocytosed into the cell and utilized. The first objective was to create an animation, involving 2D, 3D, and protein data bank based images to educate graduate students during classroom lectures. The second objective was to include a diagrammatic illustration of the entire cycle. The third objective was to determine if showing the broad and narrow views simultaneously or going back and forth between the two would better educate the audience. Two presentations were created of the LDL receptor pathway in the two styles and then evaluated by a pool of biomedical students.Item Causes of high blood cholesterol: implications for treatment(1991-02-07) Grundy, Scott M.Item Characterization of the Non-Proteolytic Mechanism and Cellular Site of Action of PCSK9-Mediated Degradation of the Low-Density Lipoprotein Receptor(2010-05-14) McNutt, Markey Carden, II; Horton, Jay D.Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a serine protease that has emerged as a central regulator of plasma low-density lipoprotein cholesterol levels. Here, it is demonstrated that PCSK9 is secreted into the blood and that the secreted PCSK9 binds and degrades LDLRs in liver. To determine if PCSK9 catalytic activity was involved in the degradation of the LDLR, a mutant PCSK9 was engineered that is catalytically inactive. Studies with catalytically inactive PCSK9 demonstrated that the protein degraded LDLRs in a manner that was indistinguishable from wild-type PCSK9, suggesting that proteolytic activity is not required for PCSK9-mediated degradation of the LDLR. Crystallographic analysis of a PCSK9:LDLR complex supported the experimental findings that PCSK9 does not catalytically cleave LDLRs. These studies also suggested that one therapeutic approach for treating hypercholesterolemia might be to disrupt the PCSK9:LDLR interaction at the cell surface. Recombinant LDLR subfragments were synthesized and added to the medium of cells that overexpressed PCSK9. These sub-fragments restored LDLRs to levels found in the control cells. These experiments confirmed that the disruption of PCSK9:LDLR at the cell surface can inhibit PCSK9 activity and suggested that the majority of PCSK9 activity is extracellular. Further structural analysis of the PCSK9:LDLR co-crystal predicted that an LDLR mutation (His306Tyr) might increase the affinity of PCSK9 for the LDLR, and thus could be associated with familial hypercholesterolemia (FH). A search of the LDLR mutation database revealed that the LDLR mutation (His306Tyr) had been reported in a kindred with FH. Structure/function studies with the mutant LDLR(H306Y) protein showed that this mutation mimics a conformation change in the wild-type LDLR that occurs at low pH, which results in increased binding. The increased affinity between PCSK9 and LDLR(H306Y) promoted enhanced LDLR degradation. Thus, this mutation represents a previously unrecognized class (Class VI) of FH mutants. Finally, two high-throughput assays were developed to discover new small molecule inhibitors of intracellular PCSK9 autocleavage and to identify previously unrecognized protein activators of PCSK9 action. Use of these assays could provide additional avenues for modulating PCSK9 activity and lead to new therapeutic options for the treatment of hypercholesterolemia.Item Development and Analysis of an Animal Model of Autosomal Recessive Hypercholesterolemia(2007-05-22) Jones, Christopher Eric Granger; Hobbs, Helen H.The low density lipoprotein receptor (LDLR) in the liver is the major route of removal of LDL cholesterol (LDL-C) from the blood. Defects in LDLR cause familial hypercholesterolemia (FH), which is characterized by elevated LDL-C, premature coronary atherosclerosis, and autosomal dominant inheritance. A similar clinical picture with an autosomal recessive mode of inheritance occurs in autosomal recessive hypercholesterolemia (ARH). ARH is caused by mutations in the ARH gene, which encodes for a putative adaptor protein implicated in linking the LDLR to the endocytic machinery. To determine the role of ARH in LDLR function, mice that do not express ARH were developed via targeted gene disruption. Similarly to ARH patients, Arh + mice have fractional catabolic rates (FCRs) for LDL similar Ldlr + mice, yet have much less severe elevations in LDL-C when fed normal chow. Upon cholesterol feeding, however, plasma lipoprotein profiles between Ldlr + and Arh + were indistinguishable. Immunolocalization studies reveal normal sorting but defective internalization of LDLRs in the livers of Arh + mice. Whereas the clearance of LDL was markedly and similarly delayed in the Arh+ and Ldlr+ mice, the rate of removal of VLDL was significantly higher in Arh + mice compared to Ldlr + animals. Primary hepatocytes expressing human LDLRs rapidly accumulated fluorescently labeled !-VLDL, but failed to internalize labeled LDL, monoclonal anti-LDLR antibody, or antibody:Protein A tetramers in the absence of ARH. These findings indicate ARH is caused by delayed clearance of LDL by LDLRs in the liver, but the severity of the disease is ameliorated by preserved VLDL clearance in the absence ARH. Thus the lower levels of LDL-C in ARH compared to FH are due to reduced production of LDL from VLDL.Item [UT News](1987-04-24) Bosler, Tommy JoyItem [UT Southwestern Medical Center News](2009-04-17) Shear, Kristen Holland