Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Structure of a Tc holotoxin pore provides insights into the translocation mechanism.

Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the 30-kDa toxic enzyme (C terminus of TcC). Binding of Tc toxins to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. A high-resolution structure of a holotoxin embedded in membranes is missing, leaving open the question of whether TcB-TcC has an influence on the conformational transition of TcA. Here we show in atomic detail a fully assembled 1.7-MDa Tc holotoxin complex from Photorhabdus luminescens in the membrane. We find that the 5 TcA protomers conformationally adapt to fit around the cocoon during the prepore-to-pore transition. The architecture of the Tc toxin complex allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Importantly, assembly of the holotoxin at the membrane results in spontaneous translocation of the toxic enzyme, indicating that this process is not driven by a proton gradient or other energy source. Mammalian lipids with zwitterionic head groups are preferred over other lipids for the integration of Tc toxins. In a nontoxic Tc toxin variant, we can visualize part of the translocating toxic enzyme, which transiently interacts with alternating negative charges and hydrophobic stretches of the translocation channel, providing insights into the mechanism of action of Tc toxins.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.

Mechanism of Tc toxin action revealed in molecular detail.

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed ß-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

A syringe-like injection mechanism in Photorhabdus luminescens toxins.

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth.

Structure and activation mechanism of the Makes caterpillars floppy 1 toxin.

The bacterial Makes caterpillars floppy 1 (Mcf1) toxin promotes apoptosis in insects, leading to loss of body turgor and death. The molecular mechanism underlying Mcf1 intoxication is poorly understood. Here, we present the cryo-EM structure of Mcf1 from Photorhabdus luminescens, revealing a seahorse-like shape with a head and tail. While the three head domains contain two effectors, as well as an activator-binding domain (ABD) and an autoprotease, the tail consists of two putative translocation and three putative receptor-binding domains. Rearrangement of the tail moves the C-terminus away from the ABD and allows binding of the host cell ADP-ribosylation factor 3, inducing conformational changes that position the cleavage site closer to the protease. This distinct activation mechanism that is based on a hook-loop interaction results in three autocleavage reactions and the release of two toxic effectors. Unexpectedly, the BH3-like domain containing ABD is not an active effector. Our findings allow us to understand key steps of Mcf1 intoxication at the molecular level.

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments.

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying Pi release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why Pi escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated Pi release and filaments with drastically shortened ADP-Pi caps. Our results provide the molecular basis for Pi release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament.

RAB33B recruits the ATG16L1 complex to the phagophore via a noncanonical RAB binding protein.

Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation.Abbreviations: ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle's balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5'-[ß,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5'-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type.

Small molecule modulation of the Drosophila Slo channel elucidated by cryo-EM.

Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca2+ concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca2+-bound and Ca2+-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K+ transport by blocking the Ca2+-induced activation signal and precludes K+ from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K+ coordination and uncouples ion gating from Ca2+ and voltage sensing. Our results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.

Compartmentalization of proteins in lipid droplet biogenesis.

Our existing understanding of the structure, protein organization and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. The electron microscopic technique of freeze-fracture replica immunogold labeling (FRIL) overcomes these problems, and is currently providing new perspectives in the field. Because of the property of frozen lipids to deflect the fracture plane, en face views of the lipid droplet and its component layers are revealed for high resolution visualization. By means of immunogold labeling, proteins involved in the accretion and mobilization of lipids, notably the PAT family proteins, can be localized at and in the droplet. Application of this approach demonstrates that, contrary to prevailing wisdom, the PAT family proteins are not invariably restricted to the surface of the lipid droplet but can occur throughout the core. The notion that lipid droplet biogenesis involves neutral lipid accumulation within the ER membrane bilayer followed by budding off, enclosed by a protein-containing phospholipid monolayer, is not substantiated. Instead, lipid droplets appear to develop externally to both ER membranes at specialized sites in which the ER enwraps the droplet, and the facing leaflets of the ER membrane and droplet surface are enriched in adipophilin. PAT family proteins are not, as often stated, specific to the lipid droplet, but are widely present in the plasma membrane where, under conditions of lipid loading, they adopt a similar configuration to that of specialized sites in the ER. FRIL has further provided new insights into the mechanism of secretion of a special type of lipid droplet, the milk fat globule. These examples highlight the contribution of the FRIL technique to critical appraisal and development of concepts in the lipid droplet field.

TIP47, a lipid cargo protein involved in macrophage triglyceride metabolism.

OBJECTIVE: Uptake of lipids by macrophages (MPhi) leads to lipid droplet accumulation and foam cell formation. The PAT family proteins are implicated in lipid droplet formation, but the precise function of the 47-kDa tail interacting protein (TIP47), a member of this family, is poorly defined. The present study was performed to determine the function of TIP47 in MPhi lipid metabolism. METHODS AND RESULTS: Freeze-fracture cytochemistry demonstrates that TIP47 is present in the plasma membrane of MPhi and is aggregated into clusters when the cells are incubated with oleate. Suppression of adipophilin levels using siRNA knockdown leads to migration of TIP47 from a cytoplasmic pool to the lipid droplet. Further, reduction of TIP47 decreases triglyceride levels, whereas raising TIP47 levels by expression of EGFP-TIP47 shows the opposite effect. CONCLUSION: Our results show that the TIP47 protein levels directly correlate with triglyceride levels. We propose that TIP47 may act as a carrier protein for free fatty acids and in this way participates in conversion of MPhi into foam cells.

Structure of the human BBSome core complex.

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine.

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.

Function of scavenger receptor class A type I/II is not important for smooth muscle foam cell formation.

Macrophages (MPhi) and smooth muscle cells (SMC) are transformed into foam cells by massive accumulation of modified lipoproteins during atherogenesis. It is known that class AI/II scavenger receptors participate in the foam cell formation of MPhi. The mechanism of lipid accumulation in SMC is however unknown. Therefore, we investigated if class AI/II scavenger receptors mediate the uptake of modified lipoproteins in SMC. Additionally, we examined the influence of MPhi and proinflammatory cytokines in this process. Our flow cytometric experiments revealed significant uptake of DiI-AcLDL in SMC. This uptake was markedly enhanced by IL-1alpha and TNF-alpha, whereas cocultured MPhi decreased the uptake of DiI-AcLDL in SMC. Competition and blocking experiments were performed to enlighten the role of class AI/II scavenger receptors. The competition experiments showed that surplus NatLDL, a ligand not known to interact with class AI/II scavenger receptors, caused a drastically decreased uptake of DiI-AcLDL in SMC. Additionally, blocking of class AI/II scavenger receptors with antibody 2F8 did not influence the uptake of DiI-AcLDL in SMC. Furthermore, fluorescence microscopic double staining of human coronary arteries with early, intermediate and advanced atherosclerotic lesions showed no colocalization of class AI scavenger receptors with SMC. These results indicate that class AI/II scavenger receptors play only a minor role in the uptake of modified lipoproteins in SMC. We suggest that SMC foam cell formation is mainly mediated by other receptors than class AI/II scavenger receptors.

Statins and foam cell formation: impact on LDL oxidation and uptake of oxidized lipoproteins via scavenger receptors.

The uptake of oxidized lipoproteins via scavenger receptors and the ensuing formation of foam cells are key events during atherogenesis. Foam cell formation can be reduced by treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). The efficacy of statins is evidently due not only to their cholesterol-lowering properties, but also to lipid-independent pleiotropic effects. This review focuses on lipid-independent pleiotropic effects of statins that influence foam cell formation during atherogenesis, with special emphasis on oxidative pathways and scavenger receptor expression.

Electron cryo-microscopy structure of the canonical TRPC4 ion channel.

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 Å. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

Pravastatin inhibits expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in Watanabe heritable hyperlipidemic rabbits: a new pleiotropic effect of statins.

BACKGROUND: LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mphis) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS AND RESULTS: Pravastatin downregulated LOX-1 expression in cultured human Mphis and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg(-1) d(-1) pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mphis and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. CONCLUSIONS: The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.