Identification of Clusterin as a Major ABri- and ADan-Binding Protein Using Affinity Chromatography.

Affinity chromatography has, for many years, been at the research forefront as one of the simplest although highly versatile techniques capable of identifying biologically relevant protein-protein interactions. In the field of amyloid disorders, the use of ligands immobilized to a variety of affinity matrices was the method of choice to individualize proteins with affinity for soluble circulating forms of amyloid subunits. The methodology has also played an important role in the identification of proteins that interact with different amyloidogenic peptides and, as a result, are capable of modulating their physiological and pathological functions by altering solubility, aggregation propensity, and fibril formation proclivity. Along this line, classical studies conducted in the field of Alzheimer's disease (AD) identified clusterin as a major binding protein to both circulating soluble Aß as well as to the brain deposited counterpart. The affinity chromatography-based approach employed herein, individualized clusterin as the major protein capable of binding the amyloid subunits associated with familial British and Danish dementias, two non-Aß neurodegenerative conditions also exhibiting cerebral amyloid deposition and sharing striking similarities to AD. The data demonstrate that clusterin binding ability to amyloid molecules is not restricted to Aß, suggesting a modulating effect on the aggregation/fibrillization propensity of the amyloidogenic peptides that is consistent with its known chaperone activity.

Clusterin in Alzheimer's disease: An amyloidogenic inhibitor of amyloid formation?

Clusterin is a heterodimeric glycoprotein (α- and ß-chain), which has been described as an extracellular molecular chaperone. In humans, clusterin is an amyloid-associated protein, co-localizing with fibrillar deposits in several amyloidoses, including Alzheimer's disease. To clarify its potential implication in amyloid formation, we located aggregation-prone regions within the sequence of clusterin α-chain, via computational methods. We had peptide-analogues, which correspond to each of these regions, chemically synthesized and experimentally demonstrated that all of them can form amyloid-like fibrils. We also provide evidence that the same peptide-analogues can inhibit amyloid-ß fibril formation, potentially making them appropriate drug candidates for Alzheimer's disease. At the same time, our findings hint that the respective aggregation-prone clusterin regions may be implicated in the molecular mechanism in which clusterin inhibits amyloid formation. Furthermore, we suggest that molecular chaperones with amyloidogenic properties might have a role in the regulation of amyloid formation, essentially acting as functional amyloids.

Glycosylated apolipoprotein J in cardiac ischaemia: molecular processing and circulating levels in patients with acute ischaemic events.

AIM: Using proteomics, we previously found that serum levels of glycosylated (Glyc) forms of apolipoprotein J (ApoJ), a cytoprotective and anti-oxidant protein, decrease in the early phase of acute myocardial infarction (AMI). We aimed to investigate: (i) ApoJ-Glyc intracellular distribution and secretion during ischaemia; (ii) the early changes in circulating ApoJ-Glyc during AMI; and (iii) associations between ApoJ-Glyc and residual ischaemic risk post-AMI. METHODS AND RESULTS: Glycosylated apolipoprotein J was investigated in: (i) cells from different organ/tissue origin; (ii) a pig model of AMI; (iii) de novo AMI patients (n = 38) at admission within the first 6 h of chest pain onset and without troponin T elevation at presentation (early AMI); (iv) ST-elevation myocardial infarction patients (n = 212) who were followed up for 6 months; and (v) a control group without any overt cardiovascular disease (n = 144). Inducing simulated ischaemia in isolated cardiac cells resulted in an increased intracellular accumulation of non-glycosylated ApoJ forms. A significant decrease in ApoJ-Glyc circulating levels was seen 15 min after ischaemia onset in pigs. Glycosylated apolipoprotein J levels showed a 45% decrease in early AMI patients compared with non-ischaemic patients (P < 0.0001), discriminating the presence of the ischaemic event (area under the curve: 0.934; P < 0.0001). ST-elevation myocardial infarction patients with lower ApoJ-Glyc levels at admission showed a higher rate of recurrent ischaemic events and mortality after 6-month follow-up (P = 0.008). CONCLUSIONS: These results indicate that ischaemia induces an intracellular accumulation of non-glycosylated ApoJ and a reduction in ApoJ-Glyc secretion. Glycosylated apolipoprotein J circulating levels are reduced very early after ischaemia onset. Its continuous decrease indicates a worsening in the evolution of the cardiac event, likely identifying patients with sustained ischaemia after AMI.

O-glycosylated clusterin as a sensitive marker for diagnosing early stages of prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) has been the most popular diagnostic marker for prostate cancer. The frequent occurrence of low PSA values (<10 ng/ml) in patients with highly suspicious prostate cancer, however, has undermined the accuracy of clinical examinations. The aim of this study was to develop a better resolution for diagnosing prostate cancer to overcome the disadvantage of PSA. METHODS: We focused on the glycosylation status of patients' serum proteins and conducted comprehensive lectin microarray analyses to characterize N- and O-glycans using sera from prostate cancer and benign prostatic diseases. Next, we retrieved candidate serum proteins with characteristic glycan structures using lectin-immobilized beads and identified them by quantitative mass spectrometry using a technique referred to as isobaric tag for relative and absolute quantitation (iTRAQ) labeling. Finally, we constructed a new assay to quantify a candidate glycoprotein with the newly identified glycans. RESULTS: Lectin microarray analyses revealed that sera from patients with prostate cancer had a higher affinity for Jacalin, Amaranthus caudatus (ACA) lectin, and Maclura pomifera (MPA) lectin, compared with that from patients with benign prostatic diseases and normal subjects, suggesting that O-glycosylated proteins are more abundant in sera from patients with prostate cancer. Then, serum glycoproteins preferentially adsorbed onto Jacalin-Agarose as well as biotin-ACA/and biotin-MPA/streptavidin-immobilized magnetic beads were isolated, labeled with iTRAQ, and identified using quantitative mass spectrometry. It was found that the ACA- and MPA-recognizable clusterin was more enriched in patients' sera from prostate cancer compared with those from benign prostatic diseases. Following this discovery, we constructed a Luminex-based assay to quantify O-glycosylated clusterin, in which total serum clusterin was first captured on anti-clusterin antibody-immobilized beads, and then clusterin-associated O-glycans were determined by the pair of biotin-MPA and streptavidin-phycoerythrin. When PSA values registered less than 10 ng/ml, the corresponding serum level of MPA-recognized clusterin determined by this assay was beneficial for distinguishing the patients with prostate cancer from the patients with benign prostatic disease. CONCLUSION: For PSA values that measure less than 10 ng/ml, the serum O-glycosylated clusterin level can be a complementary indicator for the malignancy of prostate cancer.

Glycosylation of Serum Clusterin in Wild-Type Transthyretin-Associated (ATTRwt) Amyloidosis: A Study of Disease-Associated Compositional Features Using Mass Spectrometry Analyses.

Wild-type transthyretin-associated (ATTRwt) amyloidosis is an age-related disease that causes heart failure in older adults. This disease frequently features cardiac amyloid fibril deposits that originate from dissociation of the tetrameric protein, transthyretin (TTR). Unlike hereditary TTR (ATTRm) amyloidosis, where amino acid replacements destabilize the native protein, in ATTRwt amyloidosis, amyloid-forming TTR lacks protein sequence alterations. The initiating cause of fibril formation in ATTRwt amyloidosis is unclear, and thus, it seems plausible that other factors are involved in TTR misfolding and unregulated accumulation of wild-type TTR fibrils. We believe that clusterin (CLU, UniProtKB P10909), a plasma circulating glycoprotein, plays a role in the pathobiology of ATTRwt amyloidosis. Previously, we have suggested a role for CLU in ATTRwt amyloidosis based on our studies showing that (1) CLU codeposits with non-native TTR in amyloid fibrils from ATTRwt cardiac tissue, (2) CLU interacts only with non-native (monomeric and aggregated) forms of TTR, and (3) CLU serum levels in patients with ATTRwt are significantly lower compared to healthy controls. In the present study, we provide comprehensive detail of compositional findings from mass spectrometry analyses of amino acid and glycan content of CLU purified from ATTRwt and control sera. The characterization of oligosaccharide content in serum CLU derived from patients with ATTRwt amyloidosis is novel data. Moreover, results comparing CLU oligosaccharide variations between patient and healthy controls are original and provide further evidence for the role of CLU in ATTRwt pathobiology, possibly linked to disease-specific structural features that limit the chaperoning capacity of CLU.

Low-density lipoprotein aggregation is inhibited by apolipoprotein J-derived mimetic peptide D-[113-122]apoJ.

Mimetic peptides are promising therapeutic agents for atherosclerosis prevention. A 10-residue class G* peptide from apolipoprotein J (apoJ), namely, D-[113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties. This prompted us to determine its effect on the aggregation process of low-density lipoprotein (LDL) particles, an early event in the development of atherosclerosis. LDL particles with and without [113-122]apoJ peptide were incubated at 37 °C with sphingomyelinase (SMase) or were left to aggregate spontaneously at room temperature. The aggregation process was analyzed by size-exclusion chromatography (SEC), native gradient gel electrophoresis (GGE), absorbance at 405 nm, dynamic light scattering (DLS), and transmission electronic microscopy (TEM). In addition, circular dichroism was used to determine changes in the secondary structure of apoB, and SDS-PAGE was performed to assess apoB degradation. At an equimolar ratio of [113-122]apoJ peptide to apoB-100, [113-122]apoJ inhibited both SMase-induced or spontaneous LDL aggregation. All methods showed that [113-122]apoJ retarded the progression of SMase-induced LDL aggregation at long incubation times. No effect of [113-122]apoJ on apoB secondary structure was observed. Binding experiments showed that [113-122]apoJ presents low affinity for native LDL but binds readily to LDL during the first stages of aggregation. Laurdan fluorescence experiments showed that mild aggregation of LDL resulted in looser lipid packaging, which was partially prevented by D-[113-122]apoJ. These results demonstrate that [113-122]apoJ peptide prevents SMase-induced LDL aggregation at an equimolar ratio and opens the possibility for the use of this peptide as a therapeutic tool.

Protein denaturation caused by heat inactivation detrimentally affects biomolecular corona formation and cellular uptake.

Adsorption of blood proteins to the surface of nanocarriers is known to be the critical factor influencing cellular interactions and eventually determining the successful application of nanocarriers as drug carriers in vivo. There is an increasing number of reports summarizing large data sets of all identified corona proteins. However, to date our knowledge about the multiple mechanisms mediating interactions between proteins and nanocarriers is still limited. In this study, we investigate the influence of protein structure on the adsorption process and focus on the effect of heat inactivation of serum and plasma, which is a common cell culture procedure used to inactivate the complement system. As in general routine lab procedure, heat inactivation was performed at 56 °C for 30 min in order to denature heat labile proteins. When nanocarriers were exposed to native versus heat inactivated serum, we saw that the cellular uptake by macrophages was significantly affected. These results were then correlated with an altered corona composition that depended on the treatment of the protein source. In summary, we were able to prove that the protein structure is one of the key parameters determining protein corona formation.

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins.

It is now well-established that the surface chemistry and "stealth" surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Characterization of ApoJ-reconstituted high-density lipoprotein (rHDL) nanodisc for the potential treatment of cerebral ß-amyloidosis.

Cerebral ß-amyloidosis is a major feature of Alzheimer's disease (AD), characterized by the accumulation of ß-amyloid protein (Aß) in the brain. Several studies have implicated lipid/lipoprotein metabolism in the regulation of ß-amyloidosis. In this regard, HDL (High Density Lipoprotein)-based therapies could ameliorate pathological features associated with AD. As apolipoprotein J (ApoJ) is a natural chaperone that interacts with Aß, avoiding its aggregation and toxicity, in this study we propose to prepare reconstituted rHDL-rApoJ nanoparticles by assembling phospholipids with recombinant human ApoJ (rApoJ). Hence, rHDL particles were prepared using the cholate dialysis method and characterized by N-PAGE, dynamic light scattering, circular dichroism and electron transmission microscopy. The preparation of rHDL particles showed two-sized populations with discoidal shape. Functionally, rHDL-rApoJ maintained the ability to prevent the Aß fibrillization and mediated a higher cholesterol efflux from cultured macrophages. Fluorescently-labelled rHDL-rApoJ nanoparticles were intravenously administrated in mice and their distribution over time was determined using an IVIS Xenogen® imager. It was confirmed that rHDL-rApoJ accumulated in the cranial region, especially in old transgenic mice presenting a high cerebral Aß load. In conclusion, we have standardized a reproducible protocol to produce rHDL-rApoJ nanoparticles, which may be potentially considered as a therapeutic option for ß-amyloid-related pathologies.

Exploring Amyloidogenicity of Clusterin: A Structural and Bioinformatics Analysis.

Clusterin, a multitasking glycoprotein, is a protein highly conserved amongst mammals. In humans, Clusterin is mainly a secreted protein, described as an extracellular chaperone with the capability of interacting with a broad spectrum of molecules. In neurodegenerative diseases, such as Alzheimer's disease, it is an amyloid associated protein, co-localized with fibrillar deposits in amyloid plaques in systemic or localized amyloidoses. An 'aggregation-prone' segment (NFHAMFQ) was located within the Clusterin α-chain sequence using AMYLPRED, a consensus method for the prediction of amyloid propensity, developed in our lab. This peptide was synthesized and was found to self-assemble into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, Attenuated Total Reflectance Fourier-Transform Spectroscopy and Congo red staining studies reveal. All experimental results verify that this human Clusterin peptide-analogue, possesses high aggregation potency. Additional computational analysis highlighted novel and at the same time, unexplored features of human Clusterin.

Amorphous protein aggregates stimulate plasminogen activation, leading to release of cytotoxic fragments that are clients for extracellular chaperones.

The misfolding of proteins and their accumulation in extracellular tissue compartments as insoluble amyloid or amorphous protein aggregates are a hallmark feature of many debilitating protein deposition diseases such as Alzheimer's disease, prion diseases, and type II diabetes. The plasminogen activation system is best known as an extracellular fibrinolytic system but was previously reported to also be capable of degrading amyloid fibrils. Here we show that amorphous protein aggregates interact with tissue-type plasminogen activator and plasminogen, via an exposed lysine-dependent mechanism, to efficiently generate plasmin. The insoluble aggregate-bound plasmin is shielded from inhibition by α2-antiplasmin and degrades amorphous protein aggregates to release smaller, soluble but relatively hydrophobic fragments of protein (plasmin-generated protein fragments (PGPFs)) that are cytotoxic. In vitro, both endothelial and microglial cells bound and internalized PGPFs before trafficking them to lysosomes. Clusterin and α2-macroglobulin bound to PGPFs to significantly ameliorate their toxicity. On the basis of these findings, we hypothesize that, as part of the in vivo extracellular proteostasis system, the plasminogen activation system may work synergistically with extracellular chaperones to safely clear large and otherwise pathological protein aggregates from the body.

Polyglycerol Surfmers and Surfactants for Direct and Inverse Miniemulsion.

Poly(ethylene glycol)-based surfactants are a prominent example for nonionic surfactants. Poly(glycerol) (PG) is discussed as a polyfunctional alternative, however, it is not yet used to stabilize miniemulsions. The anionic polymerization of glycidyl ethers is used to prepare surfactants for direct or inverse emulsions and ambident surfactants by adjusting the copolymer composition. Orthogonal-protected poly(glycerol) block copolymers, using ethoxyethyl glycidyl ether and allyl glycidyl ether (AGE) or tert-butyl glycidyl ether (tBuGE), are synthesized. After cleavage of the acetal groups, these all-polyglycerol surfactants (PG-b-PtBuGE) or multifunctional surfmers (PG-b-PAGE), are used in direct and inverse miniemulsion polymerizations. Polystyrene nanoparticles are obtained by free-radical miniemulsion polymerization, in which the allyl-functionalized copolymers act as surfmer. In inverse miniemulsion, hydroxyethyl starch nanocarriers are synthesized with PG-b-PAGE as surfmer, transferred into aqueous PG-b-PtBuGE solution, and functionalized by thiol-ene addition. The PG-b-PtBuGE with equal block length ratio is used as a surfactant for direct and inverse miniemulsions. With the PG being covalently bound to the nanocarriers, a desorption during protein adsorption does not occur. It is believed that these surfactants are promising alternatives to conventional surfactants with additional functionality.

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation.

Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and ß-clusterin. We have investigated the conformational features, cellular localization, lipid accumulation, in vivo effects and histological changes upon administration of recombinant individual chains of clusterin. We demonstrate that recombinant α- and ß-chains exhibit structural and functional differences and differ in their sub-cellular localization. Full-length clusterin is known to lower lipid levels. In contrast, we find that ß-chain-treated cells accumulate 2-fold more lipid than controls. Interestingly, α-chain-treated cells do not show such increase. Rabbits injected with ß-chain, but not α-chain, show ~40% increase in weight, with adipocyte hypertrophy, liver and kidney steatosis. Many, sometimes contrasting, roles are ascribed to clusterin in obesity, metabolic syndrome and related conditions. Our findings of differential localization and activities of individual chains of clusterin should help in understanding better the roles of clusterin in metabolism.

Clusterin in the protein corona plays a key role in the stealth effect of nanoparticles against phagocytes.

In biological fluids, nanoparticles interact with biological components such as proteins, and a layer called the "protein corona" forms around the nanoparticles. It is believed that the composition of the protein corona affects the cellular uptake and in vivo biodistribution of nanoparticles; however, the key proteins of the protein corona that control the biological fate of nanoparticles remain unclear. Recently, it was reported that clusterin binding to pegylated nanoparticles is important for the stealth effect of pegylated nanoparticles in phagocytes. However, the effect of clusterin on non-pegylated nanoparticles is unknown, although it is known that clusterin is present in the protein corona of non-pegylated nanoparticles. Here, we assessed the stealth effect of clusterin in the corona of non-pegylated silver nanoparticles and silica nanoparticles. We found that serum- and plasma-protein corona inhibited the cellular uptake of silver nanoparticles and silica nanoparticles in phagocytes and that the plasma-protein corona showed a greater stealth effect compared with the serum-protein corona. Clusterin was present in both the serum- and plasma-protein corona, but was present at a higher level in the plasma-protein corona than in the serum-protein corona. Clusterin binding to silver nanoparticles and silica nanoparticles suppressed the cellular uptake of nanoparticles in human macrophage-like cells (THP-1 cells). Although further studies are required to determine how clusterin suppresses non-specific cellular uptake in phagocytes, our data suggest that clusterin plays a key role in the stealth effect of not only pegylated nanoparticles but also non-pegylated nanoparticles.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 µm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

miRNA-378 reverses chemoresistance to cisplatin in lung adenocarcinoma cells by targeting secreted clusterin.

Cisplatin resistance is a major obstacle in the treatment of NSCLC, and its mechanism has not been fully elucidated. The objectives of the study were to determine the role of miR-378 in the sensitivity of lung adenocarcinoma cells to cisplatin (cDDP) and its working mechanism. With TargetScan and luciferase assay, miR-378 was found to directly target sCLU. miR-378 and sCLU were regulated in A549/cDDP and Anip973/cDDP cells to investigate the effect of miR-378 on the sensitivity and apoptotic effects of cDDP. The effect of miR-378 upregulation on tumor growth was analyzed in a nude mouse xenograft model. The correlation between miR-378 and chemoresistance was tested in patient samples. We found that upregulation of miR-378 in A549/cDDP and Anip973/cDDP cells significantly down-regulated sCLU expression, and sensitized these cells to cDDP. miR-378 overexpression inhibited tumor growth and sCLU expression in a xenograft animal model. Analysis of human lung adenocarcinoma tissues revealed that the cDDP sensitive group expressed higher levels of miR-378 and lower levels of sCLU. miR-378 and sCLU were negatively correlated. To conclude, we identified sCLU as a novel miR-378 target, and we showed that targeting sCLU via miR-378 may help disable the chemoresistance against cisplatin in lung adenocarcinoma cells.

Supported Lipid Bilayer Platform To Test Inhibitors of the Membrane Attack Complex: Insights into Biomacromolecular Assembly and Regulation.

Complement activation plays an important role in innate immune defense by triggering formation of the membrane attack complex (MAC), which is a biomacromolecular assembly that exhibits membrane-lytic activity against foreign invaders including various pathogens and biomaterials. Understanding the details of MAC structure and function has been the subject of extensive work involving bulk liposome and erythrocyte assays. However, it is difficult to characterize the mechanism of action of MAC inhibitor drug candidates using the conventional assays. To address this issue, we employ a biomimetic supported lipid bilayer platform to investigate how two MAC inhibitors, vitronectin and clusterin, interfere with MAC assembly in a sequential addition format, as monitored by the quartz crystal microbalance-dissipation (QCM-D) technique. Two experimental strategies based on modular assembly were selected, precincubation of inhibitor and C5b-7 complex before addition to the lipid bilayer or initial addition of inhibitor followed by the C5b-7 complex. The findings indicate that vitronectin inhibits membrane association of C5b-7 via a direct interaction with C5b-7 and via competitive membrane association onto the supported lipid bilayer. On the other hand, clusterin directly interacts with C5b-7 such that C5b-7 is still able to bind to the lipid bilayer, and clusterin affects the subsequent binding of other complement proteins involved in the MAC assembly. Taken together, the findings in this study outline a biomimetic approach based on supported lipid bilayers to explore the interactions between complement proteins and inhibitors, thereby offering insight into MAC assembly and regulation.