Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) ultimately lead to migration of circulating monocytes into the artery wall. Lipid-loaded monocytes proliferate and become macrophage foam cells, the hallmark of atherosclerotic lesions. A proposed mechanism for the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apoA-I) acting as a mediator of cholesterol efflux from cells and subsequent foam cell regression. To better understand the biological changes stimulated by apoA-I treatment, differential gene expression analysis of microarray data was performed on spleen cells from mice treated with recombinant HDL (rHDL). LDL receptor null (LDLr-/-) and LDL receptor and apoA-I null (LDLr-/-, apoA-I-/-) mice were fed a Western diet consisting of 0.2% cholesterol and 42% calories as fat (HF) for a total of 12 weeks. After six weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of rHDL (protein weight) three times a week for the remaining six weeks. The control group of mice was subcutaneously injected with 200 micrograms of bovine serum albumin (BSA). Spleen cell RNA was isolated, purified, and analyzed via Illumina BeadArray Microarray Technology. Individual differential gene expression analysis that contrasted treated to non-treated groups for each genotype was performed. LDLr-/-, apoA-I-/- rHDL treated mice showed 281 significantly differentially expressed genes compared to non-treated mice while LDLr-/- mice had 1502 such genes. Of the significant genes, 189 intersected across both genotypes. In LDLr-/-, apoA-I-/-, 73 of these were up-regulated and 116 were down-regulated. LDLr-/- similarly showed 71 of the intersected genes to be up-regulated and 118 to be down-regulated. One-directional gene set pathway analysis was also performed. LDLr-/-, apoA-I-/- treated mice revealed 49 significant pathways while LDLr-/- showed a total of 63. Of these, 21 were up-regulated and 14 were down-regulated in both genotypes. Of the overrepresented, up-regulated pathways, eight of the top ten most significant ones were related to immune cells. Major functions involved receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests apoA-I treatment induces similar gene expression changes across different genotypes in mouse spleen cells.

No description available.

THE TRYPANOLYTIC FACTOR OF HUMAN SERUM: A TROJAN HORSE African trypanosomes, the prototype of which is Trypanosoma brucei, are protozoan parasites of huge clinical, veterinary and economical importance. They develop in the body fluids of various mammals (including humans) where they face and manipulate many different aspects of the immune system. The extent of this interplay is pivotal to both host and parasite survival, and depending on parasite virulence and host susceptibility, infection duration ranges from some months to several years. At the end, host survival is invariably compromised. Humans and few other primates provide however a striking exception to this fatal outcome. They are indeed fully protected against most trypanosome infections through the presence in their blood of a so-called trypanosome lytic factor (TLF). The TLF is known to circulate mainly in the form of a high density lipoprotein particle characterized by the simultaneous presence of two primate-specific proteins: haptoglobin-related protein (Hpr) and apolipoprotein L-I (apoL-I). We have contributed to delineate the respective roles played by Hpr and apoL-I in the lysis process. ApoL-I was shown to be the exclusive toxin of the TLF. In its absence humans get fully susceptible to any trypanosome infection. The toxin was shown to kill the parasite after endocytosis through the generation of ionic pores in the lysosomal membrane. Those pores dissipate membrane potential and trigger the influx of chloride ions from the cytoplasm into the lysosomal compartment, leading to an eventually fatal uncontrolled osmotic phenomenon. ApoL-I efficient delivery to the parasite relies on Hpr. African trypanosomes indeed fulfil their heme nutritional requirements by receptor-mediated internalization of the complex formed by haptoglobin, an evolutionary conserved acute-phase protein, and hemoglobin, resulting from physiological intravascular hemolysis. This heme uptake by the auxotrophic parasites contributes to both growth rate and resistance against host oxidative burst. In human serum, the trypanosome receptor is unable to discriminate between Hp and the closely related TLF-bound Hpr, explaining TLF efficient endocytosis. As such, the TLF acts as a Trojan horse, killing the parasite from inside the cell after having deceived its vigilance through the high similarity between heme-delivering haptoglobin and toxin-associated Hpr.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/28). Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 92-104.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in arteries contributing to atherosclerosis. In hereditary amyloidosis (AApoAI), a potentially fatal disease, N-terminal fragments of variant apoA-I deposit in vital organs and damage them. There is no cure for apoA-I amyloidosis and its structural basis is unknown. Previously, AApoAI mutations were mapped on the crystal structure of the human C-terminally truncated Δ(185-243)apoA-I. The results suggested that the mutation-induced destabilization of the lipid-free protein initiates β-aggregation. Our biophysical studies showed that amyloidogenic mutations G26R, W50R, F71Y and L170P did not necessarily destabilize the native structure, prompting us to search for additional triggers of apoA-I misfolding. We mapped residue segments predicted to promote β-aggregation (termed amyloid hot spots) on the atomic structure of ∆(185-243)apoA-I. The results suggested that perturbed packing of these hot spots, particularly residues 14-22, triggers amyloidosis. This enabled us to propose the first molecular mechanism of apoA-I misfolding. To explore a potential mechanism, we combined structural, stability, dynamics and functional studies of several amyloidogenic mutants and a non-amyloidogenic control, L159R. All mutants reduced structural protection of the segment 14-22, supporting our hypothesis that increased dynamics of this segment triggers AApoAI. The non-amyloidogenic mutant showed helical unfolding near the mutation site indicating susceptibility to proteolysis. We propose that the major factors that make apoA-I amyloidogenic are reduced protection of the major amyloidogenic segments combined with the structural integrity of the four-helix bundle to facilitate protein aggregation. Together, our results suggest that the fate of apoA-I in vivo depends on the balance between its misfolding, proteolysis, and protective protein-lipid interactions. Our structural and bioinformatics analysis of other members of the apolipoprotein family (A-II, A-IV, A-V, B, C-I, C-II, C-III, E, SAA) showed that apolipoproteins’ propensity to form amyloid is rooted in the proteins’ hydrophobicity, which is key to the lipid binding ability. The overlap of functional and pathologic interfaces suggests competition between normal protein function and misfolding. Therefore, increasing apolipoprotein retention on the lipid surface provides a potential therapeutic strategy against amyloidosis.