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In Vivo and In Vitro Studies Support That a New Splicing Isoform of OLR1 Gene Is Protective Against Acute Myocardial Infarction

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the “non-risk” disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

LOX-1 as a natural IFN-α–mediated signal for apoptotic cell uptake and antigen presentation in dendritic cells

The identification of molecules responsible for apoptotic cell (AC) uptake by dendritic cells (DCs) and induction of T-cell immunity against AC-associated antigens is a challenge in immunology. DCs differentiated in the presence of interferon-alpha (IFN-alpha-conditioned DCs) exhibit a marked phagocytic activity and a special attitude in inducing CD8(+) T-cell response. In this study, we found marked overexpression of the scavenger receptor oxidized low-density lipoprotein receptor 1 (LOX-1) in IFN-alpha-conditioned DCs, which was associated with increased levels of genes belonging to immune response families and high competence in inducing T-cell immunity against antigens derived from allogeneic apoptotic lymphocytes. In particular, the capture of ACs by IFN-alpha DCs led to a substantial subcellular rearrangement of major histocompatibility complex class I and class II molecules, along with enhanced cross-priming of autologous CD8(+) T cells and CD4(+) T-cell activation. Remarkably, AC uptake, CD8(+) T-cell cross-priming, and, to a lesser extent, priming of CD4(+) T lymphocytes were inhibited by a neutralizing antibody to the scavenger receptor LOX-1 protein. These results unravel a novel LOX-1-dependent pathway by which IFN-alpha can, under both physiologic and pathologic conditions, render DCs fully competent for presenting AC-associated antigens for cross-priming CD8(+) effector T cells, concomitantly with CD4(+) T helper cell activation.

The splice variant LOXIN inhibits LOX-1 receptor function through hetero-oligomerization

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a central role in the pathogenesis of atherosclerosis. We have recently identified a truncated naturally occurring variant of the human receptor LOX-1, named LOXIN, which lacks part of the C-terminus lectin-like domain. In vivo and in vitro studies support that the new splicing isoform is protective against acute myocardial infarction. The mechanism by which LOXIN exerts its protective role is unknown. In this paper we report studies on the heterologous expression and functional characterization of LOXIN variant in mammalian fibroblasts and human endothelial cells. We found that LOXIN, when expressed in the absence of LOX-1, shows diminished plasma membrane localization and is deficient in ox-LDL ligand binding. When co-transfected with the full-length counterpart LOX-1, the two isoforms interact to form LOX-1 oligomers and their interaction leads to a decrease in the appearance of LOX-1 receptors in the plasma membrane and a marked impairment of ox-LDL binding and uptake. Co-immunoprecipitation studies confirmed the molecular LOX-1/LOXIN interaction and the formation of non-functional hetero-oligomers. Our studies suggest that hetero-oligomerization between naturally occurring isoforms of LOX-1 may represent a general paradigm for regulation of LOX-1 function by its variants.

Functional Analysis and Molecular Dynamics Simulation of LOX-1 K167N Polymorphism Reveal Alteration of Receptor Activity

The human lectin-like oxidized low density lipoprotein receptor 1 LOX-1, encoded by the ORL1 gene, is the major scavenger receptor for oxidized low density lipoprotein in endothelial cells. Here we report on the functional effects of a coding SNP, c.501G>C, which produces a single amino acid change (K>N at codon 167). Our study was aimed at elucidating whether the c.501G>C polymorphism changes the binding affinity of LOX-1 receptor altering its function. The presence of p.K167N mutation reduces ox-LDL binding and uptake. Ox-LDL activated extracellular signal-regulated kinases 1 and 2 (ERK 1/2) is inhibited. Furthermore, ox-LDL induced biosynthesis of LOX-1 receptors is dependent on the p.K167N variation. In human macrophages, derived from c.501G>C heterozygous individuals, the ox-LDL induced LOX-1 46 kDa band is markedly lower than in induced macrophages derived from c.501G>C controls. Investigation of p.K167N mutation through molecular dynamics simulation and electrostatic analysis suggests that the ox-LDL binding may be attributed to the coupling between the electrostatic potential distribution and the asymmetric flexibility of the basic spine residues. The N/N-LOX-1 mutant has either interrupted electrostatic potential and asymmetric fluctuations of the basic spine arginines.

Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrPSc formation

Conversion of the cellular prion protein (PrPC) into the abnormal scrapie isoform (PrPSc) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER‐retained anti‐prion recombinant single‐chain Fv fragments have been proved to be an effective tool for inhibition of PrPC trafficking to the cell surface and antagonize PrPSc formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec‐8H4) in order to compel PrPC outside the cells. The stable expression of the Sec‐8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrPC traffic from its vesicular secretion and, most importantly, a total inhibition of PrPSc accumulation in NGF‐differentiated Sec‐8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrPC is an effective strategy to inhibit PrPSc accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.

Altered intracellular distribution of PrPC and impairment of proteasome activity in tau overexpressing cortical neurons.

The microtubule associated protein tau plays a crucial role in Alzheimers disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-regulation of the proteasome system, suggesting a possible mechanism that contributes to intracellular PrP(C) accumulation. These findings open a new perspective for the possible crosstalk between tau and prion proteins in the pathogenesis of tau induced-neurodegeneration.

Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3–H4 histones and lamin-B receptor

Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1β intrabody sequesters HP1β into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1β:LBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organisation.