Spontaneous hypercholesterolemia in cynomolgus monkeys: evidence for defective low-density lipoprotein catabolism.

Spontaneously hypercholesterolemic (SH) cynomolgus monkeys were identified that have average plasma cholesterol of 202 mg/dl, while that in normal monkeys is 119 mg/dl. The LDL from these SH monkeys have lower affinity for fibroblast LDL receptors in vitro. The amount of LDL2 (1.030 mean value of d 1.063 g/ml) required to displace 50% of [125I]LDL was 3.8 micrograms/ml for normal LDL2 and 6.6 micrograms/ml for SH-LDL2. The binding affinity of LDL1 (1.019 mean value of d 1.030 g/ml) was the same in normal and SH animals. LDL turnover experiments showed that the SH monkeys were comprised of two populations. Normal LDL2 was cleared much slower in two of the SH monkeys than in normocholesterolemic animals, suggesting that these two animals have an LDL receptor defect. However, LDL2 isolated from these two SH monkeys was cleared normally in normal monkeys. LDL2 isolated from two other SH monkeys is cleared slower than is normal LDL2 in normal animals, suggesting that these animals have an LDL defect. Thus, the hypercholesterolemia of these SH monkeys is associated with defective LDL catabolism; two animals appear to have functionally defective LDL receptors, and two animals appear to have functionally defective LDL.     

Characterization of steroid hormone association with human plasma lipoproteins

Partition coefficient analysis, equilibrium dialysis, and computer simulation were used to evaluate associations of twelve steroid hormones (androstanediol, androstenediol, androstenedione, androsterone, dehydroepiandrosterone, dihydrotestosterone, estradiol, estriol, estrone, hydroxyprogesterone, progesterone, and testosterone) with human plasma high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL). It was determined that partitioning of steroid hormones (SH) between the aqueous medium and the surfaces of lipoproteins (LP) was the initial (first order) SH-LP interaction. For some SH, especially dehydroepiandrosterone, significant second order interactions, which may involve chemical conversions, were detected. The first order binding values of the twelve SH with three LP were combined with the corresponding binding values of SH with sex hormone-binding globulin, corticosteroid-binding globulin, and albumin in a 6 X 12 matrix. The computer program TRANSPORT was used to analyze the matrix and determine the distribution of each SH among six different binding agents in the "normal" male. It was concluded that LP are important vehicles for SH conveyance in plasma and may also be important for SH entry into cells.     

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation

The aim of the present study was to verify the extent of oxidative stress induced by a meal at plasma and LDL level, and to investigate the capacity of red wine to counteract this action. In two different sessions, six healthy men ate the same test meal consisting of "Milanese" meat and fried potatoes. The meal was taken either with 400 ml red wine or with an isocaloric hydroalcoholic solution. Oxidative stress at plasma level was estimated through the measure of ascorbic acid, alpha-tocopherol, protein SH groups, uric acid, and antioxidant capacity, measured before and 1 and 3 h after the meal. The change in the resistance of LDL to oxidative modification was taken as an index of exposure to pro-oxidants. The susceptibility to Cu(II)-catalyzed oxidation of baseline and postprandial LDL was measured as conjugated dienes formation, tryptophan residues, and relative electrophoretic mobility. The experimental meal taken with wine provoked a significant increase in the total plasma antioxidant capacity and in the plasma concentration of alpha-tocopherol and SH groups. Postprandial LDL was more susceptible to metal-catalyzed oxidation than the homologous baseline LDL after the ethanol meal. On the contrary, postprandial LDL obtained after the wine meal was as resistant or more resistant to lipid peroxidation than fasting LDL.     

Evidence that apoB-100 of low-density lipoproteins is a novelsrc-related protein kinase

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of thesrc protein kinase family.     

Evidence that apoB-100 of low-density lipoproteins is a novelsrc-related protein kinase

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of thesrc protein kinase family.     

Reduced cholesterol and triglycerides in mice with a mutation in Mia2, a liver protein that localizes to ER exit sites

Through forward genetic screening in the mouse, a recessive mutation (couch potato, cpto) has been discovered that dramatically reduces plasma cholesterol levels across all lipoprotein classes. The cpto mutation altered a highly conserved residue in the Src homology domain 3 (SH3) domain of the Mia2 protein. Full-length hepatic Mia2 structurally and functionally resembled the related Mia3 protein. Mia2 localized to endoplasmic reticulum (ER) exit sites, suggesting a role in guiding proteins from the ER to the Golgi. Similarly to the Mia3 protein, Mia2's cytosolic C terminus interacted directly with COPII proteins Sec23 and Sec24, whereas its lumenal SH3 domain may facilitate interactions with secretory cargo. Fractionation of plasma revealed that Mia2(cpto/cpto) mice had lower circulating VLDL, LDL, HDL, and triglycerides. Mia2 is thus a novel, hepatic, ER-to-Golgi trafficking protein that regulates cholesterol metabolism.     

Partial characterization of low density lipoprotein preparations isolated from fresh and frozen plasma after radiolabeling by seven different methods

Four 99mTc and three 123I labeling methods were evaluated for their suitability to label low density lipoproteins (LDL) for the purpose of scintigraphic biodistribution studies. For 99mTc these methods were: direct incorporation in LDL of 99mTcO4- using sodium dithionite (dithionite method); a method using first N,N-dimethylformamide to prepare a 99mTc-complex reacting with LDL in a subsequent step (DMF method); a technique in which 99mTcO4- is first coupled to a diamide dithiolate derivative of pentanoic acid by reduction with dithionite, followed by coupling of this ligand to LDL (N2S2 method); and a method using sodium borohydride and stannous chloride as reducing agents (borohydride method). The iodination techniques were based on oxidation of I(-)----I+, using iodine monochloride (ICl method), 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen method), and N-bromosuccinimide (NBS method) as oxidants. We studied labeling yields, modification of LDL caused by the labeling procedures using agarose-gel electrophoresis, and radiochemical stability of the labeled LDL complex upon incubation in plasma at 37 degrees C for 15 h. We used Sepharose CL6B chromatography to separate LDL from other plasma proteins. We also examined whether LDL isolated from frozen plasma (Pool-LDL) gave results similar to LDL obtained from freshly prepared plasma (Fresh-LDL). Pool-LDL radiolabeled by the dithionite, DMF, NBS, and Iodogen methods lost its label upon incubation with plasma. This also happened with Fresh-LDL when the DMF, NBS and Iodogen methods were used. Upon agarose-gel electrophoresis, no modification of LDL was observed with all methods when the radionuclide/LDL ratio was kept low.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

Single spin density gradient ultracentrifugation method for the detection and isolation of light and heavy low density lipoprotein subfractions

A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.     

Simple method for comparing large numbers of flow cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral blood lymphocytes

We have developed a simple method for comparing the relative fluorescence intensity (FI) of flow cytometry histograms. It entails assessment of the FI (equivalent to the fluorescence-activated cell sorter (FACS) channel) of the 50th or 75th percentiles of either positively stained cells or the total cell population. We illustrate the method with dilution curves of 1) monoclonal antibodies against the T4 surface antigen of human peripheral blood lymphocytes and 2) fluorescent low density lipoprotein (LDL) binding to the human peripheral blood lymphocytes LDL receptor. We demonstrate the versatility of the method by characterizing the binding properties of fluorescent LDL to their receptors. Binding was shown to be specific and of high affinity, and to reach a steady state plateau at about 2 hr; the affinity of fluorescent LDL for the receptor was found to be two to three times higher than that of the unlabeled LDL.     

Decoding protein-protein interactions through combinatorial chemistry: sequence specificity of SHP-1, SHP-2, and SHIP SH2 domains

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry. The results reveal that the N-terminal SH2 domain of SHP-2 is capable of recognizing four different classes of pY peptides. Binding competition studies suggest that the four classes of pY peptides all bind to the same site on the SH2 domain surface. The C-terminal SH2 domains of SHP-1 and SHP-2 and the SHIP SH2 domain each bind to pY peptides of a single consensus sequence. Database searches using the consensus sequences identified most of the known as well as many potential interacting proteins of SHP-1 and/or SHP-2. Several proteins are found to bind to the SH2 domains of SHP-1 and SHP-2 through a new, nonclassical ITIM motif, (V/I/L)XpY(M/L/F)XP, which corresponds to the class IV peptides selected from the pY library. The combinatorial library method should be generally applicable to other protein domains.     

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.     

水稻白叶枯病候选抗性基因SHNLR的RGAs克隆及分析

旨在从含有疣粒野生稻抗白叶枯病基因的新种质SH5、SH76基因组中克隆抗病基因。利用RGAs法得到1个NBS-LRR类同源基因,暂命名为SHNLR(登录号为JF934724)。结果表明,SHNLR的开放阅读框长度为3 105 bp,编码1 034个氨基酸,含有CC、NB-ARC与LRR结构域,具备CC-NBS-LRR类植物抗病基因的结构特征。BLASTn和BLASTp比对显示SHNLR是单拷贝基因,未发现同源性较高且功能已知的基因,仅NBS保守域序列与番茄Prf基因的相似度最高。对SHNLR基因电子定位,发现其位于水稻第11号染色体的长臂末端,但与11号染色体上已定位或克隆的8个白叶枯病抗性基因具有不同序列或处于不同的位置。半定量RT-PCR分析表明,SHNLR在抗病新种质叶片中的表达明显受到白叶枯病菌Zhe173的诱导。因此推测SHNLR可能是1个与抗白叶枯病相关的R基因。     

Short peptide receptor mimics for atherosclerosis risk assessment of LDL

Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum sample volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.     

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.     

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox). We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).     

Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A "one-bead-one-compound" pY peptide library is synthesized on 90-microm TentaGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B.