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Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

Secretory granules are the cellular organelles mediating storage and regulated secretion of proteins. Their biogenesis involves sorting of secretory protein cargo and membrane constituents, which takes place at two distinct levels, the trans-Golgi network and the immature secretory granule. At both levels, sorting is accomplished by cargo aggregation and cargo-membrane recognition. Given not only the aggregative properties of the regulated secretory proteins but also the ability of lipids to form distinct membrane microdomains, self-organization of both lumenal and membrane constituents is proposed to play a crucial role in secretory granule biogenesis.

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe ( i ) an approach to identify the TPST protein, referred to as MSC ( m odification after s ubstrate c rosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [ 35 S]sulfate transfer from the cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and ( ii ) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896–2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an ≈13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

Chromogranins A and B, two widespread neuroendocrine secretory proteins, contain a homologous N-terminal disulfide-bonded loop that is required for sorting to secretory granules. Here we have investigated the role of this loop in the oligomerization of chromogranin A. Reduction of the disulfide bond or the addition of an excess of an N-terminal chromogranin A fragment containing the loop (CgA1-60) resulted in the dissociation into monomers of the chromogranin A dimer found at pH 7.4 and 6.4 and of the chromogranin tetramer found at pH 5.4. The addition of an excess of a synthetic peptide corresponding to the conserved C-terminal domain of chromogranin A (CgA406-431) had no effect on the chromogranin dimers at pH 7.4 and 6.4 and resulted in the dissociation of the chromogranin A tetramers at pH 5.4 into dimers. Fluorescence energy transfer experiments using fluorescently labeled CgA1-60 showed that the N-terminal disulfide-bonded loop has a high affinity for homodimerization (KD = 20 nM at pH 6.4), which was sufficient to mediate dimerization of full-length chromogranin A. Association and dissociation of loop-mediated chromogranin A dimerization approached completion within a few seconds. Our results imply that chromogranin A homodimerizes shortly after synthesis in the endoplasmic reticulum and that the loop-mediated homodimeric state is an essential prerequisite for its sorting, in the trans-Golgi-network, to secretory granules.

For the bilinear Hilbert transform given by: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathit{H\hspace{.167em}fg}}({\mathit{x}}){\mathrm{\hspace{.167em}=\hspace{.167em}p.v.}}{\int }{\mathit{f}}({\mathit{x\hspace{.167em}-\hspace{.167em}y}}){\mathit{g}}({\mathit{x\hspace{.167em}+\hspace{.167em}y}}){\mathrm{\hspace{.167em}}}\frac{{\mathit{dy}}}{{\mathit{y}}}{\mathrm{,}}\end{equation*}\end{document} we announce the inequality ∥ H fg ∥ p 3 ≤ K p 1 , p 2 ∥ f ∥ p 1 ∥ g ∥ p 2 , provided 2 < p 1 , p 2 < ∞, 1/ p 3 = 1/ p 1 + 1/ p 2 and 1 < p 3 < 2.

A method for affinity purification based on the high-affinity interaction of biotin-streptavidin and elution by irradiation with UV light was developed. As an example of this approach a biotinylated derivative of the peptide hormone cholecystokinin (CCK-8s) containing a photocleavable o-nitrobenzylester group was synthesized. The analog retained high affinity to both CCK receptors and antiCCK antibodies. It bound to a streptavidin-agarose affinity matrix and was subsequently released by irradiation with UV light of wavelengths >320 nm. For affinity purification of specific antibodies, the modified CCK-8s was incubated with anti-CCK antiserum, and the complex was subsequently passed over a streptavidin-agarose affinity matrix. After washing, the bound antibodies were eluted by photocleavage of the affinity ligand. The eluted antibodies showed essentially unchanged binding characteristics. The approach may prove to be generally useful in the isolation of labile proteins in an intact form.

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.

Hepatic transport of the synthetic somatostatin analog octreotide-SMS 201-995, (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Throl--and its novel derivative N-alpha-(alpha-D-glucosyl(1-4)-1-deoxy-D-fructosyl)-octreotide--SD Z CO-611, N-alpha-(alpha-D-glucosyl(1-4)-1-deoxy-D-fructosyl)-(D)Phe-Cys-Phe- (D)Trp-Lys-Thr-Cys-Throl--was studied. In rats SMS 201-995 showed a plasma elimination half-life of 1.2 +/- 0.2 hr; that of SDZ CO-611 was 1.9 +/- 0.3 hours. Within 120 min 66% of a mesenterically injected 4.4-nmol dose of SMS 201-995 was excreted in bile, but only 5.3% of SDZ CO-611 was excreted in bile. Biliary concentration of SMS 201-995 showed a maximum enrichment of 540-fold +/- 75-fold over peripheral blood concentration, indicating hepatic transport mechanisms different from simple diffusion. Comparison of plasma profiles of both peptides after mesenteric and femoral administration demonstrated the relative importance of hepatic extraction for SMS 201-995 but not for SDZ CO-611. The mode of extraction was studied by means of multiple-indicator dilution in isolated perfused rat liver, with inulin as nonpermeable marker. Ratio plots, ln([inulin]/[peptide]) vs. time, exhibited decreasing slopes for SMS 201-995, suggesting very rapid binding to hepatocyte membranes. The slope of the ratio plot of (inulin/SDZ CO-611) was almost zero even at low doses (down to 0.2 microgram), implying mainly extracellular distribution and nonhepatic elimination. Binding assays indicated the absence of somatostatin receptors in sinusoidal hepatocyte membranes. However, SMS 201-995 and SDZ CO-611 bound with high affinity to somatostatin receptors in rat cortical membranes. Multiple-indicator dilution experiments in presence of increasing cholyltaurine concentrations suggested an interaction of SMS 201-995 with sinusoidal bile salt transport. In isolated hepatocytes, uptake of SMS 201-995 was saturable and showed mutual inhibition with cholyltaurine. The results indicate that SMS 201-995 transport is different from receptor mediated endocytosis as known for peptide hormones and elimination pathways of SDZ CO-611 other than biliary excretion.

In this article we study bilinear operators whose input consists of two functions on the real line and whose output is again a function on the real line. We shall be concerned with a priori estimates for these operators. For simplicity we initially require the two input functions to be Schwartz functions and allow the output function to be a tempered distribution. By abstract generalities such a bilinear operation B can be formally written as

with constants Cfi;p1;p2 depending only on fi;p1;p2 and p := p1p2 p1+p2 hold. The flrst result of this type is proved in [4], and the purpose of the current paper is to extend the range of exponents p1 and p2 for which (2) is known. In particular, the case p1 =2 ,p2 = 1 is solved to the a‐rmative. This was

A new photoreactive tritiated cholecystokinin (CCK) analogue was synthesized which contains the p-benzoylbenzoyl moiety linked to an ornithine residue at the N-terminus of the sulfated CCK octapeptide (CCK-8s). p-Benzoylbenzoyl-Orn(propionyl)-CCK-8s bound specifically and with high affinity to CCK binding sites in membranes both from pig cerebral cortex and from rat pancreatic membranes. The apparent dissociation constants KD were 1.2 nM and 0.5 nM, respectively. [3H]-p-Benzoylbenzoyl-Orn(propionyl)-CCK-8s was incubated with CCKB receptor preparations enriched by lectin chromatography and subsequently photoactivated. A polypeptide migrating with an apparent molecular weight M(r) of 56,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was specifically labeled. The labeling was suppressed by the CCKB receptor agonist pentagastrin. The efficiency of incorporation of radioactivity was high, reaching up to 70% of specifically bound radioactivity. After treatment with trifluoromethanesulfonic acid, the molecular weight of the labeled protein decreased to 45,000, indicating that the receptor is glycosylated. Photoaffinity labeling of CCKA receptors from rat pancreas resulted in the specific labeling of a protein band with M(r) of 80,000-100,000. Our synthetic approach should be useful for the design of photoreactive analogues of a variety of peptides. The high efficiency photolabeling of the CCKB receptor will be valuable for further characterization and purification of this receptor.

We previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-β-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a “pearling” state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli.

Analysis of variants in three genes encoding oxysterol-binding protein (OSBP) homologues (OSBPL2, OSBPL9, OSBPL10) in Finnish families with familial low high-density lipoprotein (HDL) levels (N = 426) or familial combined hyperlipidemia (N = 684) revealed suggestive linkage of OSBPL10 single-nucleotide polymorphisms (SNPs) with extreme end high triglyceride (TG; >90th percentile) trait. Prompted by this initial finding, we carried out association analysis in a metabolic syndrome subcohort (Genmets) of Health2000 examination survey (N = 2,138), revealing association of multiple OSBPL10 SNPs with high serum TG levels (>95th percentile). To investigate whether OSBPL10 could be the gene underlying the observed linkage and association, we carried out functional experiments in the human hepatoma cell line Huh7. Silencing of OSBPL10 increased the incorporation of [(3)H]acetate into cholesterol and both [(3)H]acetate and [(3)H]oleate into triglycerides and enhanced the accumulation of secreted apolipoprotein B100 in growth medium, suggesting that the encoded protein ORP10 suppresses hepatic lipogenesis and very-low-density lipoprotein production. ORP10 was shown to associate dynamically with microtubules, consistent with its involvement in intracellular transport or organelle positioning. The data introduces OSBPL10 as a gene whose variation may contribute to high triglyceride levels in dyslipidemic Finnish subjects and provides evidence for ORP10 as a regulator of cellular lipid metabolism., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45–84 of proPC2 (proPC245–84) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC245–84. Rather, proPC245–84 was shown to bind to protein-free liposomes in a pH- and Ca2+-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.

Background Lipids are stored within cells in lipid droplets (LDs). They consist of a core of neutral lipids surrounded by a monolayer of phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and can rapidly change in size upon lipid uptake or release. These dynamics require a fast adaptation of LD surface. We have recently shown that two Lands cycle PC synthesizing enyzmes, LPCAT1 and LPCAT2 can localize to the LD surface. Results Here, we show that knock-down of both enzymes leads to an increase in LD size without changes in the total amount of neutral lipids, while interference with the de-novo Kennedy pathway PC biosynthesis is associated with changes in triacylglyceride synthesis. We show that function of LPCAT1 and 2 is conserved in Drosophila melanogaster by the ortholog CG32699. Furthermore we demonstrate that modulation of the LD pool by LPCAT1 influences the release of lipoprotein from liver cells. Conclusion Activity of the Kennedy pathway regulates the balance between phospholipids and neutral lipids, while the Lands cycle regulates lipid droplet size by regulating surface availability and influencing surface to volume ratio. Differences in lipid droplet size may account for differences in lipid dynamics and be relevant to understand lipid overload diseases. Electronic supplementary material The online version of this article (doi:10.1186/s12860-014-0043-3) contains supplementary material, which is available to authorized users.