APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

Endothelin-converting enzyme 1 and ß-arrestins exert spatiotemporal control of substance P-induced inflammatory signals.

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins ß-arrestin (ßARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and ßARR1/2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. ßARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from ßARRs, to recycle. Thus, both ECE-1 and ßARRs mediate the resensitization of NK1R Ca(2+) signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or ßARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of ßARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation.

Proteolytic activation of the human epithelial sodium channel by trypsin IV and trypsin I involves distinct cleavage sites.

Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK(178)), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.

Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons.

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with ß-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and ß-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/ß-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from ß-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained ß-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

The bile acid receptor TGR5 does not interact with ß-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of ß-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with ß-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with ß-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-ß-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with ß-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Neurotensin-induced proinflammatory signaling in human colonocytes is regulated by ß-arrestins and endothelin-converting enzyme-1-dependent endocytosis and resensitization of neurotensin receptor 1.

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, ß-arrestin-1 (ßARR1), and ß-arrestin-2 (ßARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of ßARR1 or ßARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves ßARRs and ECE-1, respectively. Our results also indicate that ßARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S.

Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.

Endosomes: a legitimate platform for the signaling train.

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.

An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc mutation-driven intestinal tumorigenesis.

Interleukin-25 (IL-25) and group 2 innate lymphoid cells (ILC2s) defend the host against intestinal helminth infection and are associated with inappropriate allergic reactions. IL-33-activated ILC2s were previously found to augment protective tissue-specific pancreatic cancer immunity. Here, we showed that intestinal IL-25-activated ILC2s created an innate cancer-permissive microenvironment. Colorectal cancer (CRC) patients with higher tumor IL25 expression had reduced survival and increased IL-25R-expressing tumor-resident ILC2s and myeloid-derived suppressor cells (MDSCs) associated with impaired antitumor responses. Ablation of IL-25 signaling reduced tumors, virtually doubling life expectancy in an Apc mutation-driven model of spontaneous intestinal tumorigenesis. Mechanistically, IL-25 promoted intratumoral ILC2s, which sustained tumor-infiltrating MDSCs to suppress antitumor immunity. Therapeutic antibody-mediated blockade of IL-25 signaling decreased intratumoral ILC2s, MDSCs, and adenoma/adenocarcinoma while increasing antitumor adaptive T cell and interferon-γ (IFN-γ)-mediated immunity. Thus, the roles of innate epithelium-derived cytokines IL-25 and IL-33 as well as ILC2s in cancer cannot be generalized. The protumoral nature of the IL-25-ILC2 axis in CRC highlights this pathway as a potential therapeutic target against CRC.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with ß-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of ß-arrestin1 and PP2A with noninternalized NK(1)R. ß-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that ß-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping ß-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires ß-arrestin1. ECE-1 promotes this process by releasing ß-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2.

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.

The lectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease.

Scavenger receptors are membrane glycoproteins that bind diverse ligands including lipid particles, phospholipids, apoptotic cells and pathogens. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is increasingly linked to atherosclerotic plaque formation. Transgenic mouse models for LOX-1 overexpression or gene knockout suggests that LOX-1 contributes to atherosclerotic plaque formation and progression. LOX-1 activation by oxidized LDL (low-density lipoprotein) binding stimulates intracellular signalling, gene expression and production of superoxide radicals. A key question is the role of leucocyte LOX-1 in pro-atherogenic lipid particle trafficking, accumulation and signalling leading to differentiation into foam cells, necrosis and plaque development. LOX-1 expression is elevated within vascular lesions and a serum soluble LOX-1 fragment appears diagnostic of patients with acute coronary syndromes. LOX-1 is increasingly viewed as a vascular disease biomarker and a potential therapeutic target in heart attack and stroke prevention.

LOX-1 scavenger receptor mediates calcium-dependent recognition of phosphatidylserine and apoptotic cells.

The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) scavenger receptor regulates vascular responses to oxidized-low-density-lipoprotein particles implicated in atherosclerotic plaque formation. LOX-1 is closely related to C-type lectins, but the mechanism of ligand recognition is not known. Here we show that human LOX-1 recognizes a key cellular phospholipid, PS (phosphatidylserine), in a Ca2+-dependent manner, both in vitro and in cultured cells. A recombinant, folded and glycosylated LOX-1 molecule binds PS, but not other phospholipids. LOX-1 recognition of PS was maximal in the presence of millimolar Ca2+ levels. Mg2+ was unable to substitute for Ca2+ in LOX-1 binding to PS, indicating a Ca2+-specific requirement for bivalent cations. LOX-1-mediated recognition of PS-containing apoptotic bodies was dependent on Ca2+ and was decreased to background levels by bivalent-cation chelation, LOX-1-blocking antibodies or PS-containing liposomes. The LOX-1 membrane protein is thus a Ca2+-dependent phospholipid receptor, revealing novel recognition of phospholipids by mammalian lectins.

Atherosclerosis and the Lectin-like OXidized low-density lipoprotein scavenger receptor.

The Lectin-like OXidized low-density lipoprotein scavenger receptor (LOX-1) is implicated in vascular inflammation and atherosclerotic plaque initiation, progression, and destabilization. LOX-1 levels are elevated upon recognition of oxidized low-density lipoprotein, a key pro-atherogenic substance in the vasculature. Recent evidence indicates this gene product is a biomarker of inflammation and disease status. We review and assess the role of LOX-1 in atherosclerotic plaque formation, physiologic regulation, and as a biomarker and target in cardiovascular disease diagnosis and prevention.

Biochemistry and cell biology of mammalian scavenger receptors.

Scavenger receptors are integral membrane proteins that bind a wide variety of ligands including modified or oxidised low-density lipoproteins, apoptotic cells and pathogens. Modified low-density lipoprotein accumulation is thought to be an early event in vascular disease and thus scavenger receptor function is critical in this context. The scavenger receptor family has at least eight different subclasses (A-H) which bear little sequence homology to each other but recognize common ligands. Here we review our current understanding of the scavenger receptor subclasses with emphasis on their genetics, protein structure, biochemical properties, membrane trafficking, intracellular signalling and links to disease states. We also highlight emerging areas where scavenger receptors play roles in cell and animal physiology.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling.

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Oxidised LDL internalisation by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and is regulated by dynamin-2.

The LOX-1 scavenger receptor recognises pro-atherogenic oxidised low-density lipoprotein (OxLDL) particles and is implicated in atherosclerotic plaque formation, but this mechanism is not well understood. Here we show evidence for a novel clathrin-independent and cytosolic-signal-dependent pathway that regulates LOX-1-mediated OxLDL internalisation. Cell surface labelling in the absence or presence of OxLDL ligand showed that LOX-1 is constitutively internalised from the plasma membrane and its half-life is not altered upon ligand binding and trafficking. We show that LOX-1-mediated OxLDL uptake is disrupted by overexpression of dominant-negative dynamin-2 but unaffected by CHC17 or mu2 (AP2) depletion. Site-directed mutagenesis revealed a conserved and novel cytoplasmic tripeptide motif (DDL) that regulates LOX-1-mediated endocytosis of OxLDL. Taken together, these findings indicate that LOX-1 is internalised by a clathrin-independent and dynamin-2-dependent pathway and is thus likely to mediate OxLDL trafficking in vascular tissues.

Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds.

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial alpha-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.