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Apolipoprotein L-I (apoL1) is a human-specific serum protein that kills Trypanosoma brucei through ionic pore formation in endosomal membranes of the parasite. The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. In the case of T. b. rhodesiense, resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1. We undertook a mutational and deletional analysis of the C-terminal helix of apoL1 to investigate the linkage between interaction with SRA and lytic potential for different T. brucei subspecies. We confirm that the C-terminal helix is the SRA-interacting domain. Although in E. coli this domain was dispensable for ionic pore-forming activity, its interaction with SRA resulted in inhibition of this activity. Different mutations affecting the C-terminal helix reduced the interaction of apoL1 with SRA. However, mutants in the L370-L392 leucine zipper also lost in vitro trypanolytic activity. Truncating and/or mutating the C-terminal sequence of human apoL1 like that of apoL1-like sequences of Papio anubis resulted in both loss of interaction with SRA and acquired ability to efficiently kill human serum-resistant T. b. rhodesiense parasites, in vitro as well as in transgenic mice. These findings demonstrate that SRA interaction with the C-terminal helix of apoL1 inhibits its pore-forming activity and determines resistance of T. b. rhodesiense to human serum. In addition, they provide a possible explanation for the ability of Papio serum to kill T. b. rhodesiense, and offer a perspective to generate transgenic cattle resistant to both T. b. brucei and T. b. rhodesiense.

Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

Liposomes are versatile carriers that can encapsulate various drugs; however, for delivery to the brain, they must be modified with a targeting ligand or other modifications to provide blood-brain barrier (BBB) permeability, while avoiding rapid clearance by reticuloendothelial systems through polyethylene glycol (PEG) modification. BBB-penetrating peptides act as brain-targeting ligands. In this study, to achieve efficient brain delivery of liposomes, we screened the functionality of eight BBB-penetrating peptides reported previously, based on high-throughput quantitative evaluation methods with in vitro BBB permeability evaluation system using Transwell, in situ brain perfusion system, and others. For apolipoprotein E mimetic tandem dimer peptide (ApoEdp), which showed the best brain-targeting and BBB permeability in the comparative evaluation of eight peptides, its lipid conjugate with serine-glycine (SG) 5 spacer (ApoEdp-SG-lipid) was newly synthesized and ApoEdp-modified PEGylated liposomes were prepared. ApoEdp-modified PEGylated liposomes were effectively associated with human brain capillary endothelial cells via the ApoEdp sequence and permeated the membrane in an in vitro BBB model. Moreover, ApoEdp-modified PEGylated liposomes accumulated in the brain 3.9-fold higher than PEGylated liposomes in mice. In addition, the ability of ApoEdp-modified PEGylated liposomes to localize beyond the BBB into the brain parenchyma in mice was demonstrated via three-dimensional imaging with tissue clearing. These results suggest that ApoEdp-SG-lipid modification is an effective approach for endowing PEGylated liposomes with the brain-targeting ability and BBB permeability.

Significance African trypanosomes are parasites that can cause African sleeping sickness in humans. Host defense against some of these is provided by the human serum factor apolipoprotein L-1 (APOL1), which causes swelling and lysis of susceptible trypanosomes. Lysis follows uptake of APOL1 into acidic parasite endosomes and is thought to involve ion influx across the plasma membrane. In this paper we show that, after interaction of APOL1 with lipid bilayers at acidic pH, subsequent pH neutralization triggers the opening of pH-gated channels that selectively conduct cations across the bilayer. Based on these results, we propose a mechanism of trypanosome lysis that involves endocytic recycling of APOL1 and the opening of APOL1-induced cation-selective channels, at neutral pH, in the parasite plasma membrane. Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei , but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense , which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pK ₐ = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cation-selective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane.

Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood–brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained increased. Likewise, intracranial administration of Aβ to apoE-targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4 > apoE3 > apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain.

Background Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. Methods Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. Results A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. Conclusions These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Background High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved. Methods Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed. Results The addition of HDL +ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL -ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex. Conclusions Altogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.

Caveola-located scavenger receptor type B class I (SR-BI) and activin receptor-like kinase-1 (ALK1) are involved in transendothelial transport of apolipoprotein B-carrying lipoproteins (apoB-LPs). Transport of apoB-LPs though mouse aortic endothelial cells (MAECs) is associated with apoE-carrying high-density lipoprotein (HDL)-like particle formation and apoAI induces raft-located proteins to shift to non-raft membranes by upregulation of ATP-binding cassette transporter A1 (ABCA1). To investigate apoAI’s effect on transendothelial transport of apoB-LPs, MAECs and human coronary artery endothelial cells (HCAECs) were treated with apoB-LPs ± apoAI. Our data demonstrated that apoAI neither altered SR-BI and ALK1 expression nor affected apoB-LP binding to MAECs. ApoAI inhibited MAEC uptake, transcellular transport, and intracellular accumulation of apoB-LPs and accelerated their resecretion in MAECs. ApoAI enhanced transendothelial apoB-LP transport-associated HDL-like particle formation, upregulated ABCA1 expression, shifted SR-BI and ALK1 to the non-raft membrane in MAECs, inhibited transcellular transport of apoB-LPs, and enhanced associated HDL-like particle formation in HCAECs. ABCA1 knockdown attenuated apoAI-induced membrane SR-BI and ALK1 relocation and diminished apoAI’s effect on transendothelial apoB-LP transport and HDL-like particle formation in MAECs. This suggests that upregulation of ABCA1 expression is a mechanism, whereby apoAI provokes caveola-located receptor relocation, inhibits transendothelial apoB-LP transport, and promotes associated HDL-like particle formation.

Background: While apolipoprotein E4 (apoE4) is highly correlated with the development of Alzheimer’s disease (AD), its role in AD pathology and, in particular, beta-amyloid (Aβ) removal from the brain, is not clearly defined. Objective: To elucidate the influence of apoE on the clearance of Aβ across the blood-brain barrier (BBB). Methods: Aβ(1–42) was intracerebrally administered to transgenic mice expressing human apoE isoforms and examined in the periphery. Results: apoE3 and apoE4 mice had 5 times and 2 times, respectively, more Aβ(1–42) appearing in the plasma than wild-type or apoE knockout mice, indicating an enhanced clearance of Aβ from the brain to the periphery. In vitro, unbound basolateral apoE3 (i.e., not bound to Aβ), and to a lesser extent unbound apoE4, at concentrations ≤10 n m facilitated basolateral-to-apical fluorescein-Aβ(1–42) transcytosis across a BBB model, while apoE isoforms bound to Aβ significantly disrupted Aβ transcytosis. Additionally, following apical exposure to the BBB model, we found that apoE4 bound to Aβ is able to penetrate the BBB more readily than apoE3 bound to Aβ and does so via the RAGE (receptor for advanced glycation end products) transporter. Conclusion: These studies indicate a multifaceted, isoform-dependent role for apoE in the exchange of Aβ across the BBB and may partially explain the association of apoE4 and Aβ brain accumulation in AD.

The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1 – 4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei , but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense , T.b. gambiense and the animal pathogens Trypanosoma evansi , Trypanosoma congolense and Trypanosoma vivax . However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi . This trypanolytic potential was partially shared by rAPOL1 from Papio papio (r Pp APOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental r Pp APOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs. Recombinant proteins based on APOL1 and APOL3 can kill pathogenic Trypanosoma brucei subspecies, including a variant (rPpMUT) that is effective against T.b. gambiense infection in mice, suggesting that it may serve as a therapy against sleeping sickness.