Effects of estradiol on very low density lipoprotein receptor mRNA levels in rabbit heart

AbstractThe VLDL receptor, a newly identified lipoprotein receptor, has a domain structure homologous with the LDL receptor but has one additional cysteine rich repeat in the ligand binding domain. To study the regulation of the VLDL receptor in vivo, we administered 17 α-ethinyl estradiol to rabbits and examined its effects on VLDL receptor mRNA levels in the heart, one of the organs with highly expressed VLDL receptors, by RNA blotting. The ventricular level of VLDL receptor mRNA increased dramatically after estradiol administration. We could not detect VLDL receptor mRNA in the liver even after estradiol administration. We have confirmed the enhanced expression of liver LDL receptor mRNA by estradiol, however, only weak expression of the LDL receptor was detected in the ventricle of the rabbit heart. These results suggest that estradiol exerts its effect on the VLDL receptor gene expression in the heart

A "de novo" mutation of the LDL-receptor gene as the cause of familial hypercholesterolemia

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Mutational analysis of ligand - LDL-receptor interactions

Lipoproteinrezeptoren sind Plasmamembranproteine, welche für die spezifische Bindung und Aufnahme der im Blutkreislauf zirkulierenden Lipoproteine in hepatische und extrahepatische Gewebe verantwortlich sind. Dieser physiologische Vorgang wird als rezeptorvermittelte Endozytose bezeichnet und basiert auf der Interaktion zwischen Mitgliedern der sogenannten Low Density Lipoprotein (LDL) Rezeptor Familie und Lipoproteinen im Plasma. Der als erster entdeckte und am besten charakterisierte Lipoproteinrezeptor ist der LDL Rezeptor (LDLR). Dieser Rezeptor spielt eine wichtige Rolle in der Regulation der Cholesterinspiegel durch die Bindung und Internalisierung von Lipoproteinen, welche Apolipoprotein- B (apoB) und /oder -E (apoE) als Proteinkomponente besitzen. In der vorliegenden Arbeit wurde das legende Huhn, ein gut etablierter Modellorganismus zur Erforschung des Lipidstoffwechsels, herangezogen um die Interaktion des LDL Rezeptors mit Apolipoprotein B- und -E hältigen Lipoproteinen näher zu charakterisieren. Früheren Studien zufolge gibt es zahlreiche Oberflächenrezeptoren im Huhn, die eine deutliche Homologie zur Familie der LDL Rezeptorproteine von Säugern aufweisen. Der zum menschlichen LDL Rezeptor homologe Rezeptor im Huhn wurde erstmals im Jahr 2003 beschrieben und weist die klassischen Strukturmerkmale der Säuger-LDL Rezeptoren auf, was auf eine hohe evolutionäre Konservierung deutet. Alle Mitglieder der LDL Rezeptorfamilie besitzen einen charakteristischen modularen Aufbau aus vier bis fünf unterschiedlichen Domänen. Die amino-terminale Region enthält die Ligandenbindungsdomäne des Rezeptors und besteht aus mehreren cysteinreichen Abschnitten, den sogenannten LA-repeats, welche die Interaktion mit verschiedenen Proteinen, wie Apolipoprotein, Proteasen und Signalproteinen vermitteln. Mutationsanalysen zeigen, dass die einzelnen Repeats unterschiedliche funktionelle Bedeutungen bei der Ligandenbindung besitzen. Bindungsstudien im Hühnersystem ergaben, dass einige Rezeptoren der LDLR Familie Apolipoprotein E binden, ein in Säugern, jedoch nicht im Huhn produziertes Protein. Andere Rezeptoren wiederum besitzen diese Bindungseigenschaft nicht. Dieses unterschiedliche Verhalten weist darauf hin, dass bestimmte Variationen in der ligandenbindenden Domäne des Rezeptors einen entscheidenden Einfluss auf die Ligandenbindung besitzen. In der vorliegenden Arbeit habe ich mich damit beschäftigt, die strukturellen Unterschiede zwischen ApoE-bindenden Rezeptoren und Rezeptoren, welche die Fähigkeit ApoE zu binden nicht besitzen, zu ermitteln. Gemäß den Ergebnissen aus früheren Mutationsanalysen wurde die Ligandenbindungsdomäne des Hühner- LDLR an vermeintlich wichtigen Bindungsregionen mutiert. Die bakteriell exprimierten mutanten Rezeptorproteine wurden durch Affninitätschromatographie gereinigt und, um die physiologische Funktionsweise zu erlangen, einem Faltungsprozess unterzogen, welcher durch das molekulare Chaperon RAP (Receptor-associated Protein) unterstützt wurde. Schließlich wurden die Rezeptormutanten hinsichtlich der Ligandenbindung von Apolipoprotein B- und -E -hältigen Lipoproteinen untersucht. Diese Bindungsanalysen ergaben, dass das erste cysteinreiche LA Repeat (LA1) nicht an der Binding von Apolipoprotein B beteiligt scheint. Indessen führten Mutationen in den Repeats 4 und 5 (LA4, LA5), sowie in der Linker-Region zwischen diesen Repeats zu erheblichen Einschränkung bezüglich der Bindung von ApoB-hältigen Lipoproteinen. Bindungsanalysen zur Erforschung der Interaktion zwischen ApoE und den Rezeptormutanten lieferten bisher keine reproduzierbaren Ergebnisse, und erfordern daher weitere Untersuchungen. Die Daten, die im Zuge dieser Arbeit durch Experimente im Huhn ermittelt wurden, tragen zum Verständnis des molekularen Mechanismus der LDL Rezeptor-Ligand Interaktion bei und stehen im Einklang mit Untersuchungsergebnissen am menschlichen LDL Rezeptor, welche eine große Bedeutung des fünften LA Repeats (LA5) für die Ligandenbindung beschreiben.Lipoprotein receptors are cell surface-exposed receptors that are specific for the binding and uptake of lipoproteins from the circulation into hepatic and extrahepatic tissues. This process is termed receptor-mediated endocytosis and is largely dependent upon the interaction between members of the so-called Low Density Lipoprotein (LDL) receptor family and plasma lipoproteins. The best characterized receptor of this family is the LDL receptor (LDLR). It plays a major role in the regulation of plasma cholesterol levels by mediating the binding and uptake of apolipoprotein B- (apoB) and -E (apoE) -containing plasma lipoprotein particles. To gain further insight into the molecular mechanism of LDL receptor- ligand interactions, I performed the present study in the domesticated chicken (Gallus gallus), which serves as an excellent model organism for research on lipid metabolism. Various surface receptors homologous to members of the mammalian LDLR family have been discovered in this species, and are recognized as important in the maintenance of systemic lipid homeostasis and embryongenesis including follicle development and oocyte growth. In 2003, the first avian LDL receptor ortholog was characterized and revealed a high degree of conservation during evolution, since the LDLR’s hallmark properties are already present in the chicken protein. The numerous avian and mammalian relatives of the receptor family share characteristic similarities, both in structural and functional terms. Via highly conserved cysteine-rich repeat elements in the extracellular ligand-binding domain the receptors are able to bind many unrelated proteins, such as apolipoproteins, proteases, signaling molecules, and several other groups of proteins. Acting as cargo transporters, members of this receptor family are not only capable of transporting macromolecules, but also of signal transduction. According to previous studies, certain chicken receptors belonging to the LDLR family are able to bind apolipoprotein E, a protein of mammalian origin, which is not produced in the avian species, while other receptors like the chicken LDLR do not have binding affinity for apoE. In agreement with previous investigations, this observation indicates that certain variations in the ligand-binding domain of the receptor contribute to the recognition of a variety of heterogeneous ligands. In the present study I focused on the investigation of structural differences between apoE-binding competent and apoE-binding incompetent receptors that are pivotal for ligand-binding. To address these issues I generated receptor fragments of the chicken LDLR consisting of the ligand-binding region. Based on previous findings derived from extensive studies of the human LDLR, mutations in putatively important receptor domains that were characterized as critical for apoE binding were introduced. Following bacterial expression, the receptor mutants were purified using affinity chromatography. Since the functionality of the LDLR depends on the correct folding including disulfide bond formation, the main challenge was to re-fold the recombinant receptor fragments into a binding-active conformation. This was achieved by the assistance of the receptor-associated protein (RAP), a molecular chaperone especially acting on LDLR family members. The refolding procedure was performed in the presence of RAP in an environment that allows disulfide bond formation and Ca2+ incorporation. Finally, the mutated receptors were characterized with regards to the ligand-binding properties towards apolipoprotein B and -E containing lipid particles. The data obtained from solid phase binding assays revealed that the first LA repeat is not required to bind apoB- containing lipoproteins. However, mutations in the linker region separating repeat 4 and 5 as well as mutations in LA repeat 5 dramatically impaired the binding of apoB-containing lipoproteins. Initial investigations examining the binding activity of the mutant receptors towards apoE-containing particles did not lead to reproducible results and thus require further investigations. These data obtained in an oviparous species contribute to the understanding of LDLR-ligand interactions and are in agreement with previous investigations, in which the importance of LA5 for ligand recognition of the human LDLR was established

Post-translational processing of the low density lipoprotein receptor

The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein that mediates the uptake of plasma LDL and thereby provides cholesterol to cells. During its synthesis in the endoplasmic reticulum, the LDL receptor folds and forms disulfide bonds in multiple cysteine-rich repeats. N- and 0-linked oligosaccharide chains are added in the endoplasmic reticulum and processed during passage through the Golgi apparatus, en route to the cell surface. The aim of this thesis was to study the influence of post-translational events on the synthesis of the LDL receptor. Experiments addressed: 1) the necessity of the compartmental organisation of the secretory pathway for the glycosylation of the LDL receptor; 2) the requirements for the formation of disulfide bonds; 3) the role for the chaperone, calnexin, in the folding of the LDL receptor; and 4) the manner in which folding was disrupted by mutations. Experiments were performed in cultured cells that were incubated with [³⁵S]methionine. Biosynthetically-labelled LDL receptor was immunoprecipitated and was analysed by SOS polyacrylamide gel electrophoresis

Molecular Markers of Tumor Progression in Melanoma

Malignant melanoma represents one of the most aggressive malignancies but outcome is highly variable with early tumor lesions having an excellent prognosis following resection. We review here the data on identification of genes involved in the progression of melanoma as a result of expression array studies, genomic profiling, and genetic models. We focus on the role of tumor suppressors involved in cell cycle function, DNA repair, and genome maintenance. Highlighted are the roles of loss of p16 in promoting neoplasia in cooperation with deregulated MAPK signaling, and the role of loss of the RASSF1A protein in promoting chromosomal instability. The interactions between point mutation in growth signaling molecules and epigenetic changes in genes involved in DNA repair and cell division are discussed

Role of the β-propeller domain of low-density lipoprotein receptor in human rhinovirus infection

Drei Vertreter der low-density Lipoprotein Rezeptor Familie, LDLR selbst, very-LDLR, und LDLR related protein (LRP) werden von minor group Rhinoviren des Menschen (HRVs) als Rezeptoren verwendet. Während ICAM-1, der Rezeptor der major group der HRVs, aktiv am viralen uncoating beteiligt ist, wurden minor group Rezeptoren als passive Vehikel betrachtet, die das Virus in Endosomen transportieren, wo sie ein Milieu mit niedrigem pH vorfinden. Für die YWTD β-Propeller Domaine des LDL-Rezeptors wurde jedoch gezeigt, dass sie an der Abdissoziation gebundenen LDLs bei niedrigem pH beteiligt ist. Wir wollten daher herausfinden, ob sie auch aktiv an der Infektion mit HRVs teilnimmt. Es existiert keine humane Zell Linie, die LDLR in Abwesenheit von VLDLR oder LRP exprimiert. Daher wurden in unseren Untersuchungen CHO-ldla7 Zellen verwendet, die keinen endogenen LDL-Rezeptor exprimieren. Diese waren stabil transfiziert um entweder Wildtyp oder β-Propeller negative humane LDL-Rezeptoren zu exprimieren. Mit Hilfe dieser Zellen untersuchten wir den möglichen Einfluss des β-Propellers auf die Konversion von HRV2 in subvirale Partikel als eine Funktion des niedrigen pH und die Freisetzung des Virus vom Rezeptor. Wenn HRV2 an propeller-negativen LDLR gebunden war ein niedrigerer pH wurde für die Konversion benötigt. Dies deutet darauf hin, dass die hoch-avide Rezeptorbindung das Virus in seiner nativen Konformation stabilisiert. Infektionsversuche zeigten eine zeitliche Verzögerung in der intrazellulären viralen Konversion, im Abbau und in der viralen de novo Synthese in der Abwesenheit der β-Propeller-Domaine. Wir schließen daraus, dass die Funktion des β-Propellers bei niedrigem pH auch dann signifikant ist, wenn der Ligand ein Rhinovirus ist. Er erleichtert die virale Konversion im Inneren von Endosomen, indem er den Virus-stabilisierenden Effekt des mit hoher Avidität bindenden LDLR reduziert. Dadurch begünstigt er die Infektion.Three members of the low-density lipoprotein receptor (LDLR) family, LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP) act as minor group human rhinovirus (HRV) receptors. Whereas ICAM-1, the receptor of the major group of HRVs, actively contributes to viral uncoating, minor group receptors were rather considered passive vehicles just delivering virus to the low pH environment of endosomes. However, the YWTDβ-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL at acidic pH. We thus aimed to clarify whether it also actively contributes to HRV infection. No human cell line expressing LDLR in the absence of VLDLR or LRP is available. Therefore, CHO-ldla7 cells which are deficient in endogenous LDLR and are stably transfected to express either wild-type or β-propeller negative human LDLR were used to investigate low pH-triggered conversion of HRV2 and its release from the receptor. Lower pH was required for conversion when HRV2 was attached to the propeller-negative LDLR, indicating that the high-avidity receptor-binding stabilizes the virus in its native conformation. Infection assays showed a delay in intracellular viral conversion, degradation, as well as in de novo viral synthesis in the absence of the β-propeller domain. We conclude that the function of the β-propeller at low pH is also significant when the ligand is a rhinovirus; it facilitates viral conversion within endosomes by decreasing the virus-stabilizing effect of the high avidity LDLR binding, and thereby promotes infection

A Surgical Perspective on Targeted Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide, is difficult to treat and highly lethal. Since HCC is predominantly diagnosed in patients with cirrhosis, treatment planning must consider both the severity of liver disease and tumor burden. To minimize the impact to the patient while treating the tumor, techniques have been developed to target HCC. Anatomical targeting by surgical resection or locoregional therapies is generally reserved for patients with preserved liver function and minimal to moderate tumor burden. Patients with decompensated cirrhosis and small tumors are optimal candidates for liver transplantation, which offers the best chance of long-term survival. Yet, only 20%-30% of patients have disease amenable to anatomical targeting. For the majority of patients with advanced HCC, chemotherapy is used to target the tumor biology. Despite these treatment options, the five-year survival of patients in the United States with HCC is only 16%. In this review we provide a comprehensive overview of current approaches to target HCC. We also discuss emerging diagnostic and prognostic biomarkers, novel therapeutic targets identified by recent genomic profiling studies, and potential applications of immunotherapy in the treatment of HCC

Adenovirus-mediated correction of the genetic defect in hepatocytes from patients with familial hypercholesterolemia

Familial hypercholesterolemia (FH) is an inherited deficiency of LDL receptors that has been an important model for liver-directed gene therapy. We are developing approaches for treating FH that are based on direct delivery of recombinant LDL receptor genes to liver in vivo. As a first step towards this goal, replication-defective recombinant adenoviruses were constructed which contained either the lacZ gene or the human LDL receptor cDNA expressed from a β-actin promoter. Primary cultures of hepatocytes were established from two patients with homozygous FH and one nonFH patient, and subsequently exposed to recombinant adenoviruses at MOIs ranging from 0.1 to 5. Essentially all of the cells expressed high levels of the transgene without demonstrable expression of an early or late adenoviral gene product; the level of recombinant-derived LDL receptor protein in transduced FH hepatocytes exceeded the endogenous levels by at least 20-fold. These studies support the utility of recombinant adenoviruses for efficient transduction of recombinant LDL receptor genes into human FH hepatocytes without expression of viral proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45545/1/11188_2005_Article_BF01233250.pd

The characterisation of the ectodomain shedding of the low density lipoprotein receptor

Includes abstract. Includes bibliographical references (leaves 142-155)