Denis Sviridov

High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of polypeptide abundance.

BACKGROUND Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass. CONTENT We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 micromol/L on a molar basis or from about 50,000 to 1 mg/L mass abundance. CONCLUSIONS Data on the concentrations of the high-abundance peptide chains in plasma assist in understanding the composition of plasma and potential approaches for clinical laboratory or proteomic analysis of plasma proteins. Development of more extensive databases regarding the plasma concentrations of proteins in health and diseases would promote diagnostic and proteomic advances.

5A Apolipoprotein Mimetic Peptide Promotes Cholesterol Efflux and Reduces Atherosclerosis in Mice

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

The dynamic range problem in the analysis of the plasma proteome.

One of the greatest challenges in analyzing the plasma proteome is the wide range of concentration of different proteins. The current study examines the range of protein concentration for 18 proteins measured over a year in a clinical laboratory to provide data on pathological extremes in protein concentrations. The complete measured range, from upper limits for albumin to lowest values for thyroid-stimulating hormone (TSH), represented more than 10 logs of molar abundance. A number of plasma proteins measured in the clinical laboratory varied over a concentration range spanning more than 4 logs, and limits of detection of clinical assays were inadequate to assess full concentration ranges of several proteins. Considering reported values from studies using higher sensitivity assays suggest that plasma concentrations of prostate-specific antigen (PSA), human chorionic gonadotropin (hCG), and cardiac troponin I vary by more than 7 logs. All of the plasma proteins measured in the present study represent secretory proteins or highly expressed components of specific tissues. Thus, the dynamic range for these components is likely to greatly underestimate the total range of protein concentration in the plasma proteome.

Diagnostic potential for urinary proteomics

Urine represents a modified ultrafiltrate of plasma, with protein concentrations typically approximately 1000-fold lower than plasma. Urines low protein concentration might suggest it to be a less promising diagnostic specimen than plasma. However, urine can be obtained noninvasively and tests of many urinary proteins are well-established in clinical practice. Proteomic technologies expand opportunities to analyze urinary proteins, identifying more than 1000 proteins and peptides in urine. Urine offers a sampling of most plasma proteins, with increased proportions of low-molecular-weight protein and peptide components. Urine also offers enriched sampling of proteins released along the urinary tract. Although urine presents some challenges as a diagnostic specimen, its diverse range of potential markers offers great potential for diagnosis of both systemic and kidney diseases. Examples of clinical situations where this may be of value are for more sensitive detection of kidney transplant rejection or of renal toxicity of medications.

Class B Scavenger Receptor Types I and II and CD36 Targeting Improves Sepsis Survival and Acute Outcomes in Mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II– or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50–70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II–deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II– and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

Comparison of Urinary Albumin Quantification by Immunoturbidimetry, Competitive Immunoassay, and Protein-Cleavage Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Increased urinary albumin excretion is a well-documented diagnostic and prognostic biomarker for renal disease. Urinary albumin is typically measured in clinical settings by immunoassay methods. However, neither a reference method nor a urine albumin calibration reference material is currently available. METHODS We quantified urinary albumin in patient samples by using 3 commercially available reagent systems: DiaSorin SPQ and Beckman Coulter LX 20 (immunoturbidimetric), and Siemens Immulite (competitive immunoassay). Results were compared to values obtained by protein-cleavage liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS In general, results from the 3 immunoassays agreed with results from LC-MS/MS. However, the SPQ results showed a negative bias across all ranges of albuminuria [(0-200 mg/L, y = 0.91x - 3.74 (CI 0.86-0.96); > 200 mg/L, y = 0.88x - 40.30 (CI 0.76-1.00)], whereas the LX 20 showed minimal bias in the 0-200 mg/L range [y = 0.97x - 88 (CI 0.92-1.02)] and the Immulite assay showed positive bias in the 0-200 mg/L range [y = 1.15x - 4.38 (CI 1.09-1.20)]. CONCLUSIONS These results showed a reasonable quantification of urinary albumin by representative polyclonal and monoclonal immunoassays compared to an LC-MS/MS assay. In addition, the results do not suggest the presence of nonimmunoreactive albumin in urine. However, differences in analytic performance between assays support the need for a reference calibration material and reference method to standardize clinical laboratory measurements of urinary albumin.

A novel apolipoprotein C-II mimetic peptide that activates lipoprotein lipase and decreases serum triglycerides in apolipoprotein E-knockout mice.

Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E–knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia.

Urine albumin measurement: Effects of urine matrix constituents

BACKGROUND There is increasing clinical interest in accurate measurement of urine albumin as an early indicator of kidney disease. However, urine is a highly variable sample matrix that may exert matrix effects on assays for urine albumin. METHODS Variation in urine composition was assessed for components routinely measured in the clinical laboratory over 1 year. Solutions of representing different concentrations of urine components were prepared and spiked with a constant amount of albumin. Fourteen urine specimens of variable composition were ultrafiltered and spiked with a constant amount of albumin. Concentrations of albumin in solutions were determined with an immunoturbidimetric assay on the Beckman LX20 analyzer and with a competitive immunoassay on the Siemens Immulite analyzer. RESULTS Variation in concentrations of most constituents altered measured urine by <10%. High NaCl concentration increased assay values by 10% or more. Addition of 0.5% Triton X-100 increased values in the LX20 assay by about 20%. Most ultrafiltered urine specimens had <10% effect on measured albumin at a concentration near 20 mg/l, but one specimen was noted to decrease measured albumin by approximately 20% in the LX20 assay. CONCLUSIONS Urine matrix components exert small but potentially significant effects on assays for urine albumin. Urine matrix effects should be considered in the design and evaluation of assays for albumin.

Increased cholesterol efflux capacity in metabolic syndrome: Relation with qualitative alterations in HDL and LCAT.

BACKGROUND Metabolic syndrome (MetS) is associated with changes in HDL levels, composition and sub-fraction profile. Whether these alterations affect HDL anti-atherogenic function, specifically measured as its capacity to perform cholesterol efflux, is not yet clearly known. OBJECTIVE To evaluate the relation between serum cholesterol efflux capacity and the changes in HDL composition and sub-fraction profile in MetS. METHODS In 35 non-treated MetS patients and 15 healthy controls, HDL mediated cholesterol efflux was measured as the ability of apoB-depleted serum to accept cholesterol from cholesterol-loaded BHK cells expressing either ABCA1 or ABCG1. Additionally we determined: lipid profile, HDL sub-fractions (NMR) and LCAT mass (ELISA). Isolated HDL (δ:1.063-1.210 g/mL) was chemically characterized. Pre-β1-HDL was determined by 2D-electrophoresis in a sub-group of MetS and controls (n = 6 each). RESULTS Surprisingly, MetS patients presented higher ABCA1 mediated cholesterol efflux (10.4 ± 1.8 vs. 8.7 ± 0.3%; p = 0.0001), without differences in ABCG1 efflux. In MetS, HDL showed reduction in particle size and number (p < 0.02) and lower large/small HDL ratio (p = 0.05), as well as triglyceride enrichment (p = 0.0001). Pre-β1-HDL was increased in MetS (p = 0.048) and correlated with ABCA1-cholesterol efflux (r = 0.64; p = 0.042). LCAT mass showed a tendency to reduction in MetS (p = 0.08), and inversely correlated with ABCA1-cholesterol efflux (r = -0.51; p = 0.001), independently of obesity and insulin-resistance (β = -0.40, p = 0.034). CONCLUSION This is the first description of ABCA1 mediated cholesterol efflux in MetS. Regardless the reduced HDL-cholesterol, in vitro cholesterol efflux capacity by ABCA1 was enhanced, linked to increased pre-β1-HDL and slightly reduced in LCAT mass that would probably reflect a delay in reverse cholesterol transport occurring in MetS.