Systematic evaluation of lecithin:cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: regulatory role of charged residues at positions 4 and 7.

Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and ß-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT ß-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.

Poly (N-vinylpyrrolidone) modification mitigates plasma protein corona formation on phosphomolybdate-based nanoparticles.

Phosphomolybdate-based nanoparticles (PMo12-based NPs) have been commonly applied in nanomedicine. However, upon contact with biofluids, proteins are quickly adsorbed onto the NPs surface to form a protein corona, which induces the opsonization and facilitates the rapid clearance of the NPs by macrophage uptake. Herein, we introduce a family of structurally homologous PMo12-based NPs (CDS-PMo12@PVPx(x = 0 ~ 1) NPs) capping diverse content of zwitterionic polymer poly (N-vinylpyrrolidone) (PVP) to regulate the protein corona formation on PMo12-based NPs. The fluorescence quenching data indicate that the introduction of PVP effectively reduces the number of binding sites of proteins on PMo12-based NPs. Molecular docking simulations results show that the contact surface area and binding energy of proteins to CDS-PMo12@PVP1 NPs are smaller than the CDS-PMo12@PVP0 NPs. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further applied to analyze and quantify the compositions of the human plasma corona formation on CDS-PMo12@PVPx(x = 0 ~ 1) NPs. The number of plasma protein groups adsorption on CDS-PMo12@PVP1 NPs, compared to CDS-PMo12@PVP0 NPs, decreases from 372 to 271. In addition, 76 differentially adsorption proteins are identified between CDS-PMo12@PVP0 and CDS-PMo12@PVP1 NPs, in which apolipoprotein is up-regulated in CDS-PMo12@PVP1 NPs. The apolipoprotein adsorption onto the NPs is proposed to have dysoponic activity and enhance the circulation time of NPs. Our findings demonstrate that PVP grafting on PMo12-based NPs is a promising strategy to improve the anti-biofouling property for PMo12-based nanodrug design.

Host Defence Cryptides from Human Apolipoproteins: Applications in Medicinal Chemistry.

Several eukaryotic proteins with defined physiological roles may act as precursors of cryptic bioactive peptides released upon protein cleavage by the host and/or bacterial proteases. Based on this, the term "cryptome" has been used to define the unique portion of the proteome encompassing proteins with the ability to generate bioactive peptides (cryptides) and proteins (crypteins) upon proteolytic cleavage. Hence, the cryptome represents a source of peptides with potential pharmacological interest. Among eukaryotic precursor proteins, human apolipoproteins play an important role, since promising bioactive peptides have been identified and characterized from apolipoproteins E, B, and A-I sequences. Human apolipoproteins derived peptides have been shown to exhibit antibacterial, anti-biofilm, antiviral, anti-inflammatory, anti-atherogenic, antioxidant, or anticancer activities in in vitro assays and, in some cases, also in in vivo experiments on animal models. The most interesting Host Defence Peptides (HDPs) identified thus far in human apolipoproteins are described here with a focus on their biological activities applicable to biomedicine. Altogether, reported evidence clearly indicates that cryptic peptides represent promising templates for the generation of new drugs and therapeutics against infectious diseases.

The role of apolipoprotein- and vitronectin-enriched protein corona on lipid nanoparticles for in vivo targeted delivery and transfection of oligonucleotides in murine tumor models.

The application of lipid-based nanoparticle (LNP) delivery systems remains a popular strategy for the systemic delivery of gene therapies to specific disease targets, including solid tumors. It is now well acknowledged that upon systemic administration, biomolecules from blood will adsorb onto nanoparticles' surfaces, forming a "protein corona", affording nanoparticles a "biological identity" on top of their "synthetic identity". Detailed analysis of nanoparticle protein corona is gradually revealing the "missing link" between nanoparticle chemical properties and the biological identity. Nevertheless, the discovery of nanoparticle protein corona's impact on tumor delivery is limited. In this study, we demonstrate that protein corona can be manipulated by formulation composition and particle surface charge changes, and a single lipid switch could switch the nanoparticle protein corona profile. The protein corona composition differences had a profound impact on cell transfection, in vivo biodistribution as well as tumor-specific delivery efficiency. Nanoparticles with apolipoprotein-rich corona showed better delivery to hepatocellular carcinoma (HepG2) as compared to those with vitronectin-rich corona. In addition, we found that, the PEG conjugated lipid chain length and PEG amount in LNPs were key factors to consider in successful RNA interference therapy for solid tumors.

Nuts and Bolts of Protein Quantification by Online Trypsin Digestion Coupled LC-MS/MS Analysis.

Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing.

Interaction of a model apolipoprotein, apoLp-III, with an oil-phospholipid interface.

Lipid droplets are "small" organelles that play an important role in de novo synthesis of new membrane, and steroid hormones, as well as in energy storage. The way proteins interact specifically with the oil-(phospho-)lipid monolayer interface of lipid droplets is a relatively unexplored but crucial question. Here, we use our home built liquid droplet tensiometer to mimic intracellular lipid droplets and study protein-lipid interactions at this interface. As model neutral lipid binding protein, we use apoLp-III, an amphipathic α-helix bundle protein. This domain is also found in proteins from the perilipin family and in apoE. Protein binding to the monolayer is studied by the decrease in the oil/water surface tension. Previous work used POPC (one of the major lipids found on lipid droplets) to form the phospholipid monolayer on the triolein surface. Here we expand this work by incorporating other lipids with different physico-chemical properties to study the effect of charge and lipid head-group size. This study sheds light on the affinity of this important protein domain to interact with lipids.

Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system.

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

Double positivity of the IgG isotype of both anticardiolipin and anti-ß2gpI antibodies is associated with the highest number of vascular impairment parameters in patients with primary antiphospholipid syndrome: preliminary data.

Although numerous studies investigated the association between homocysteine (Hcy), tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP) and apolipoproteins (apos) with thrombosis and/or recurrent pregnancy losses, studies that analyzed the abovementioned parameters and multiple positivity of antiphospholipid antibodies (aPL Abs) in patients with primary antiphospholipid syndrome (PAPS) are lacking. Therefore, the aim of this study was to analyze the presence of various combinations of the abovementioned parameters and their associations with clinical and/or serological features of PAPS. High-pressure liquid chromatography (HPLC) was used for determination of Hcy, while apoAI, apoB, and lipoprotein (Lp) (a) concentrations were estimated by immunonephelometry. High-sensitivity C-reactive protein (hsCRP) was measured by immunoturbidimetry. Apo (a), TNF-α, and aPL Abs were measured by ELISA. Various combinations of analyzed parameters (Hcy/CRP/TNF/apoAI/apoB/apo (a)/Lp (a)) were not associated with a single presence of either aPL Abs. Double positivity for both isotypes of anticardiolipin (aCL) Abs (IgG + IgM) was associated with the increased apoB levels. The presence of the IgG isotype of both aCL + anti-beta2 glycoprotein I (aß2gpI) Abs was associated with the highest number of analyzed parameters (i.e., increased levels of hsCRP, Lp (a), and apo (a)). The presence of the IgG isotype of both aCL + aß2gpI Abs was associated with the highest number of vascular impairment parameters in patients with PAPS, and this combination confers the highest risk for the recurrence of thrombotic episodes. This is the first report that analyzed the association between various combinations of vascular impairment parameters with multiple aPL Ab positivity. Our results provide a rationale for further investigations of therapeutic approaches for PAPS patients.

Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.

A magnetic-dependent protein corona of tailor-made superparamagnetic iron oxides alters their biological behaviors.

In recent years, it is becoming increasingly evident that once nanoparticles come into contact with biological fluids, a protein corona surely forms and critically affects the biological behaviors of nanoparticles. Herein, we investigate whether the formation of protein corona on the surface of superparamagnetic iron oxides (SPIOs) is influenced by static magnetic field. Under static magnetic field, there is no obvious variation in the total amount of protein adsorption, but the proportion of adsorbed proteins significantly changes. Noticeably, certain proteins including apolipoproteins, complement system proteins and acute phase proteins, increase in the protein corona of SPIOs in the magnetic field. More importantly, the magnetic-dependent protein corona of SPIOs enhances the cellular uptake of SPIOs into the normal cell line (3T3 cells) and tumor cell line (HepG2 cells), due to increased adsorption of apolipoprotein. In addition, SPIOs with the magnetic-dependent protein corona cause high cytotoxicity to 3T3 cells and HepG2 cells. This work discloses that superparamagnetism as a key feature of SPIOs affects the composition of protein corona to a large extent, which further alters the biological behaviors of SPIOs.

Amyloid-Forming Properties of Human Apolipoproteins: Sequence Analyses and Structural Insights.

Apolipoproteins are protein constituents of lipoproteins that transport cholesterol and fat in circulation and are central to cardiovascular health and disease. Soluble apolipoproteins can transiently dissociate from the lipoprotein surface in a labile free form that can misfold, potentially leading to amyloid disease. Misfolding of apoA-I, apoA-II, and serum amyloid A (SAA) causes systemic amyloidoses, apoE4 is a critical risk factor in Alzheimer's disease, and apolipoprotein misfolding is also implicated in cardiovascular disease. To explain why apolipoproteins are over-represented in amyloidoses, it was proposed that the amphipathic α-helices, which form the lipid surface-binding motif in this protein family, have high amyloid-forming propensity. Here, we use 12 sequence-based bioinformatics approaches to assess amyloid-forming potential of human apolipoproteins and to identify segments that are likely to initiate ß-aggregation. Mapping such segments on the available atomic structures of apolipoproteins helps explain why some of them readily form amyloid while others do not. Our analysis shows that nearly all amyloidogenic segments: (i) are largely hydrophobic, (ii) are located in the lipid-binding amphipathic α-helices in the native structures of soluble apolipoproteins, (iii) are predicted in both native α-helices and ß-sheets in the insoluble apoB, and (iv) are predicted to form parallel in-register ß-sheet in amyloid. Most of these predictions have been verified experimentally for apoC-II, apoA-I, apoA-II and SAA. Surprisingly, the rank order of the amino acid sequence propensity to form amyloid (apoB>apoA-II>apoC-II≥apoA-I, apoC-III, SAA, apoC-I>apoA-IV, apoA-V, apoE) does not correlate with the proteins' involvement in amyloidosis. Rather, it correlates directly with the strength of the protein-lipid association, which increases with increasing protein hydrophobicity. Therefore, the lipid surface-binding function and the amyloid-forming propensity are both rooted in apolipoproteins' hydrophobicity, suggesting that functional constraints make it difficult to completely eliminate pathogenic apolipoprotein misfolding. We propose that apolipoproteins have evolved protective mechanisms against misfolding, such as the sequestration of the amyloidogenic segments via the native protein-lipid and protein-protein interactions involving amphipathic α-helices and, in case of apoB, ß-sheets.

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells.

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper-ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.

Apolipoprotein M: Research progress, regulation and metabolic functions (Review).

Apolipoprotein M (ApoM) is a novel lipoprotein-associated plasma protein of the apolipoprotein family. It is predominantly enriched in high-density lipoprotein (HDL), and is also present in small quantities in low-density lipoprotein (LDL) and in very low-density lipoprotein. Transgenic animal experiments have suggested that ApoM can be transformed into various lipoproteins and may be involved in lipoprotein metabolism. ApoM has five subtypes, however, their biological functions remain to be elucidated. The α-helix, formed by ApoM through hydrophobic signal peptides, is anchored to the phospholipid monomolecular layers of HDL. Hydrophobic domains can associate with small lipophilic ligands and perform biological functions. ApoM may affect HDL metabolism and exhibit anti-atherosclerotic functions. Human HDL, containing ApoM subfractions, can protect LDL from oxidation and regulate cholesterol efflux more effectively than HDL without ApoM. Therefore, it is highly correlated with plasma cholesterol levels in the human body. Although previous studies have reported no difference in ApoM between groups of patients with coronary heart disease (CHD) and a normal control groups, the anti-atherosclerotic effect of ApoM is evident. ApoM is highly expressed in renal proximal tubule cells and is secreted into the urine in tubule cells. However, it is usually reabsorbed by giantin-associated proteins in a process, which is also affected in kidney disease. In addition to liver and kidney cells, low expression levels of ApoM occur in the intestinal tract and are associated with lymph node metastasis of colorectal cancer. ApoM gene polymorphism is associated with CHD, diabetes and other immune-associated diseases. Investigations into the gene regulation of ApoM may assist in further clarifying the role of ApoM in blood glucose and lipid metabolism. Genetic modification of the mouse ApoM gene is an essential technique to investigate the gene expression and regulation of ApoM, and to clarify the potential roles of ApoM in lipoprotein metabolism, atherosclerosis, diabetes and renal diseases.

Uncleaved ApoM signal peptide is required for formation of large ApoM/sphingosine 1-phosphate (S1P)-enriched HDL particles.

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM(Q22A)) introduces a functional signal peptidase cleavage site. Expression of apoM(Q22A) in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM(WT)). When apoM(Q22A) was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM(WT). Hepatocytes isolated from both apoM(WT)- and apoM(Q22A)-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM(WT), apoM(Q22A) hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM(Q22A) bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM(WT) and apoM(Q22A) hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.

A retained secretory signal peptide mediates high density lipoprotein (HDL) assembly and function of haptoglobin-related protein.

Haptoglobin-related protein (Hpr) is a component of a minor subspecies of high density lipoproteins (HDL) that function in innate immunity. Here we show that assembly of Hpr into HDL is mediated by its retained N-terminal signal peptide, an unusual feature for a secreted protein and the major difference between Hpr and the soluble acute phase protein haptoglobin (Hp). The 18-amino acid signal peptide is necessary for binding to HDL and interacts directly with the hydrocarbon region of lipids. Utilizing model liposomes, we show that the rate of assembly and steady-state distribution of Hpr in lipid particles is mediated by the physical property of lipid fluidity. Dye release assays reveal that Hpr interacts more rapidly with fluid liposomes. Conversely, steady-state binding assays indicate that more rigid lipid compositions stabilize Hpr association. Lipid association also plays a role in facilitating hemoglobin binding by Hpr. Our data may offer an explanation for the distinct distribution of Hpr among HDL subspecies. Rather than protein-protein interactions mediating localization, direct interaction with phospholipids and sensitivity to lipid fluidity may be sufficient for localization of Hpr and may represent a mechanism of HDL subspeciation.

The human HDL proteome displays high inter-individual variability and is altered dynamically in response to angioplasty-induced atheroma plaque rupture.

Recent findings support potential roles for HDL in cardiovascular pathophysiology not related to lipid metabolism. We address whether HDL proteome is dynamically altered in atheroma plaque rupture. We used immunoaffinity purification of HDL samples from coronary artery disease patients before and after percutaneous transluminal coronary angioplasty (PTCA), a model of atheroma plaque disruption. Samples were analyzed by quantitative proteomics using stable isotope labeling and results were subjected to statistical analysis of protein variance using a novel algorithm. We observed high protein variability in HDL composition between individuals, indicating that HDL protein composition is highly patient-specific. However, intra-individual protein variances remained at low levels, confirming the reproducibility of the method used for HDL isolation and protein quantification. A systems biology analysis of HDL protein alterations induced by PTCA revealed an increase in two protein clusters that included several apolipoproteins, fibrinogen-like protein 1 and other intracellular proteins, and a decrease in antithrombin-III, annexin A1 and several immunoglobulins. Our results support the concept of HDL as dynamic platforms that donate and receive a variety of molecules and provide an improved methodology to use HDL proteome for the systematic analysis of differences among individuals and the search for cardiovascular biomarkers. Biological significance The HDL proteome is an interesting model of clinical relevance and has been previously described to be dynamically altered in response to pathophysiological conditions and cardiovascular diseases. Our study suggests that interindividual variability of HDL proteome is higher than previously thought and provided the detection of a set of proteins that changed their abundance in response to plaque rupture, supporting the concept of HDL as dynamic platforms that donate and receive a variety of molecules.

Human LDL structural diversity studied by IR spectroscopy.

Lipoproteins are responsible for cholesterol traffic in humans. Low density lipoprotein (LDL) delivers cholesterol from liver to peripheral tissues. A misleading delivery can lead to the formation of atherosclerotic plaques. LDL has a single protein, apoB-100, that binds to a specific receptor. It is known that the failure associated with a deficient protein-receptor binding leads to plaque formation. ApoB-100 is a large single lipid-associated polypeptide difficulting the study of its structure. IR spectroscopy is a technique suitable to follow the different conformational changes produced in apoB-100 because it is not affected by the size of the protein or the turbidity of the sample. We have analyzed LDL spectra of different individuals and shown that, even if there are not big structural changes, a different pattern in the intensity of the band located around 1617 cm(-1) related with strands embedded in the lipid monolayer, can be associated with a different conformational rearrangement that could affect to a protein interacting region with the receptor.

Associations between smoking, components of metabolic syndrome and lipoprotein particle size.

BACKGROUND: The clustering of metabolic and cardiovascular risk factors is known as metabolic syndrome (MetS). The risk of having MetS is strongly associated with increased adiposity and can be further modified by smoking behavior. Apolipoproteins (apo) associated with low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) may be altered in MetS. This study aimed to examine the association between smoking and the following parameters: MetS and its components, levels of apolipoproteins and estimated lipoprotein particle size, separately for men and women, and in different body mass index (BMI) classes. METHODS: We included 24,389 men and 35,078 women aged between 18 and 80 years who participated in the LifeLines Cohort Study between December 2006 and January 2012; 5,685 men and 6,989 women were current smokers. Participants were categorized into three different body mass index (BMI) classes (BMI <25; BMI 25 to 30; BMI ≥30 kg/m²). MetS was defined according to the National Cholesterol Education Program's Adult Treatment Panel III (NCEP:ATPIII) criteria. Blood pressure, anthropometric and lipid measurements were rigorously standardized, and the large sample size enabled a powerful estimate of quantitative changes. The association between smoking and the individual MetS components, and apoA1 and apoB, was tested with linear regression. Logistic regression was used to examine the effect of smoking and daily tobacco smoked on risk of having MetS. All models were age adjusted and stratified by sex and BMI class. RESULTS: Prevalence of MetS increased with higher BMI levels. A total of 64% of obese men and 42% of obese women had MetS. Current smoking was associated with a higher risk of MetS in both sexes and all BMI classes (odds ratio 1.7 to 2.4 for men, 1.8 to 2.3 for women, all P values <0.001). Current smokers had lower levels of HDL cholesterol and apoA1, higher levels of triglycerides and apoB, and higher waist circumference than non-smokers (all P <0.001). Smoking had no consistent association with blood pressure or fasting blood glucose. In all BMI classes, we found a dose-dependent association of daily tobacco consumption with MetS prevalence as well as with lower levels of HDL cholesterol, higher triglyceride levels and lower ratios of HDL cholesterol/apoA1 and, only in those with BMI <30, LDL cholesterol/apoB (all P <0.001). CONCLUSIONS: Smoking is associated with an increased prevalence of MetS, independent of sex and BMI class. This increased risk is mainly related to lower HDL cholesterol, and higher triglycerides and waist circumference. In addition, smoking was associated with unfavorable changes in apoA1 and apoB, and in lipoprotein particle size. Please see related commentary: http://www.biomedcentral.com/1741-7015/11/196.

Solvent-induced α- to 3(10)-helix transition of an amphiphilic peptide.

The amphiphilic peptide of the triacylglycerol lipase derived from Pseudomonas aeruginosa plays a critical role in guarding the gate for ligand access. Conformations of this peptide at several water-oil interfaces and in protein environments were compared using atomistic simulations with explicit solvents. In oil-containing solvents, this peptide is able to retain a folded structure. Interestingly, when the peptide is immersed in a low-polarity solvent environment, it exhibits a "coalesced" helix structure, which has both α- and 3(10)-helix components. The observation that the 3(10)-helical conformation is populated in a highly nonpolar environment is consistent with a previous report on polymethylalanine. Frequent interconversions of the secondary structure (between α-helix and 3(10)-helix) of the peptide are also observed. We further studied how this solvent-induced structural transition may be connected to the trigger mechanism of lipase gating and how the lipase senses the hydrophobic-hydrophilic interface.

Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.

PURPOSE: We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of seven apolipoproteins (Apos) in human serum by LC-MS/MS using proteotypic peptides and corresponding stable isotope-labeled peptides as internal standards. EXPERIMENTAL DESIGN: Micro-LC was coupled with quadrupole-linear ion trap MS for quantification and peptide confirmation. Denaturation, reduction, alkylation, and tryptic digestion including ultrasound and microwave assistance were investigated. Method comparison of 50 plasma samples with an immunoassay was performed for Apo A-I and Apo B. RESULTS: Tryptic digestion times ranged between 5 min (Apo A-I, Apo E, Apo A-IV) and 16 h (Apo A-II). Ultrasound and microwave assistance did not improve the digestion yield. Linearity was found between 0.1 nmol/L and 100 mmol/L. The lower limits of quantification were ≤ 0.4 µmol/L for Apo A-I, Apo A-IV, Apo B-100, Apo C-I, Apo C-III, Apo E, and <1.4 µmol/L for Apo A-II. CV <13% were determined. Comparison with immunoassays showed a good agreement for Apo A-I and Apo B. CONCLUSION AND CLINICAL RELEVANCE: The validated preanalytical protocol enables a reliable simultaneous analysis of seven Apos in human serum without depletion. The method can now be applied in clinical studies to investigate the Apo distributions in cardiovascular diseases.