Biomedical subjects

Lelio Orci

Publications and source records attributed to Lelio Orci.

At least 19 recordsLinked to original sources

PubMed2006

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment.

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.

Adipose Tissue, White↗

PubMed2006

Exomer: A coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast.

A yeast plasma membrane protein, Chs3p, transits to the mother-bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)-binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPgammaS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPgammaS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.

ADP-Ribosylation Factor 1↗

PubMed2006

Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking.

Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects mapped to chromosome 14q13-q21 (ref. 1). Here we show, using a positional cloning approach, that an F382L amino acid substitution in SEC23A segregates with this syndrome. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. These cells also exhibit cytoplasmic mislocalization of SEC31. Cell-free vesicle budding assays show that the F382L substitution results in loss of SEC23A function. A phenotype reminiscent of CLSD is observed in zebrafish embryos injected with sec23a-blocking morpholinos. Our observations suggest that disrupted endoplasmic reticulum export of the secretory proteins required for normal morphogenesis accounts for CLSD.

Abnormalities, Multiple↗

PubMed2005

Fat storage in adipocytes requires inactivation of leptin's paracrine activity: implications for treatment of human obesity.

Hyperleptinemia rapidly depletes adipocyte fat in lean rats, whereas comparable hyperleptinemia produced by adipocytes in diet-induced obesity does not, implying a leptinergic blockade in adipocytes during overnutrition. Indeed, activated STAT-3 in white adipose tissue (WAT) of normal rats was less on a 60% high fat diet (HFD) than on 4% fat, despite a 10-fold higher plasma leptin. In 6 days of a HFD, mRNA of the postreceptor leptin inhibitor, suppressor of cytokine signaling-3, increased 22-fold in WAT, while leptin receptor (Lepr-b) mRNA gradually disappeared, implying leptinergic blockade at both postreceptor and receptor levels. Adipocyte-specific Lepr-b overexpression of a Lepr-b transgene completely prevented the adipocyte hypertrophy and hyperplasia and the increase in body fat induced in wild-type mice by HFD. Activated STAT-3 and AMP-activated protein kinase (AMPK), and the mRNA of lipooxidative enzymes, peroxisome proliferator-activated receptor-gamma-coactivator-1alpha, and uncoupling protein-1 and -2 were increased in WAT. Body temperature was elevated in the transgenic mice, suggesting uncoupled fatty acid oxidation of surplus fatty acids. In conclusion, storage of surplus calories in WAT and the development of diet-induced obesity require the blockade of a latent leptin-stimulated caloric sump in white adipocytes.

Adenylate Kinase↗

PubMed2005

Dynamic transport of SNARE proteins in the Golgi apparatus.

Localization of a membrane protein in a subcellular compartment can be achieved by its retention in the compartment or by its continuous transport toward this compartment. Previous results have suggested that specific enzymes are localized in the Golgi apparatus at least in part by selective retention and exclusion from transport vesicles. However, the function of some Golgi SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins is not compatible with their exclusion from transport vesicles. To help understand the mechanism accounting for the localization of SNARE proteins in the Golgi apparatus, we analyzed their lateral distribution in the Golgi cisternae and their incorporation into transport vesicles. According to our results, all SNARE proteins are efficiently incorporated into transport vesicles, indicating that the localization of SNARE proteins in the Golgi apparatus is not based on a static retention mechanism. Detailed analysis suggested that incorporation into transport vesicles was more efficient for SNARE proteins restricted to the cis face of the Golgi as compared with SNAREs present at the trans face. Furthermore, overexpression of a cis-Golgi SNARE protein altered concomitantly its incorporation in transport vesicles and its intra-Golgi localization. These observations suggest that, contrary to resident Golgi enzymes, SNARE proteins are localized in the Golgi apparatus as the result of a dynamic transport equilibrium.

Animals↗

PubMed2005

Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle.

Secretory proteins traffic from the ER to the Golgi via COPII-coated transport vesicles. The five core COPII proteins (Sar1p, Sec23/24p, and Sec13/31p) act in concert to capture cargo proteins and sculpt the ER membrane into vesicles of defined geometry. The molecular details of how the coat proteins deform the lipid bilayer into vesicles are not known. Here we show that the small GTPase Sar1p directly initiates membrane curvature during vesicle biogenesis. Upon GTP binding by Sar1p, membrane insertion of the N-terminal amphipathic alpha helix deforms synthetic liposomes into narrow tubules. Replacement of bulky hydrophobic residues in the alpha helix with alanine yields Sar1p mutants that are unable to generate highly curved membranes and are defective in vesicle formation from native ER membranes despite normal recruitment of coat and cargo proteins. Thus, the initiation of vesicle budding by Sar1p couples the generation of membrane curvature with coat-protein assembly and cargo capture.

Binding Sites↗

PubMed2005

Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

Animals↗

PubMed2004

Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles.

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.

Adenosine Triphosphate↗

PubMed2004

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes.

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.

COP-Coated Vesicles↗

PubMed2004

Rapid transformation of white adipocytes into fat-oxidizing machines.

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor gamma coactivator 1alpha (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.

Adenoviridae↗

PubMed2004

Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.

Animals↗

PubMed2004

Generation and characterization of telomerase-transfected human lymphatic endothelial cells with an extended life span.

The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.

Biomarkers↗

PubMed2004

Bi-directional protein transport between the ER and Golgi.

The endoplasmic reticulum (ER) and the Golgi comprise the first two steps in protein secretion. Vesicular carriers mediate a continuous flux of proteins and lipids between these compartments, reflecting the transport of newly synthesized proteins out of the ER and the retrieval of escaped ER residents and vesicle machinery. Anterograde and retrograde transport is mediated by distinct sets of cytosolic coat proteins, the COPII and COPI coats, respectively, which act on the membrane to capture cargo proteins into nascent vesicles. We review the mechanisms that govern coat recruitment to the membrane, cargo capture into a transport vesicle, and accurate delivery to the target organelle.

Animals↗

PubMed2003

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

Binding Sites↗

PubMed2003

Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles.

We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p. We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging of a number of cargo molecules is disrupted when mutations are introduced at this site. Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree. We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p. Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p. Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.

Amino Acid Sequence↗

PubMed2003

Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae.

Yeast endoplasmic reticulum (ER) vesicle protein Erv46p is a novel membrane protein involved in transport through the early secretory pathway. Investigation of mammalian Erv46 (mErv46) reveals that it is broadly expressed in tissues and protein-secreting cells. By immunofluorescence microscopy, mErv46 displays a crescent-shaped perinuclear staining pattern that is characteristic of the Golgi complex. Quantitative immunoelectron microscopy indicates that mErv46 is restricted to the cis face of the Golgi apparatus and to vesicular tubular structures between the transitional ER and cis-Golgi. Minor amounts of mErv46 reside in ER membranes and later Golgi cisternae. On Brefeldin A treatment, mErv46 redistributes to punctate structures that costain for ERGIC53. Depletion of mErv46 protein by RNA interference caused no apparent structural changes in the intermediate compartment or Golgi complex. These findings place mErv46 in a group of itinerant proteins that cycle between the ER and Golgi compartments such as ERGIC53 and the p24 proteins.

Animals↗

PubMed2003

Self-assembly of minimal COPII cages.

The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm. Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.

COP-Coated Vesicles↗

PubMed2002

Lipoapoptosis: its mechanism and its diseases.

The balance between cell division and cell death determines the cell population of an organ. When cell death exceeds cell replacement in an organ, a functional deficit is created. A metabolic cause of programmed cell death, lipoapoptosis, has recently been identified to occur in obesity and aging. If nonadipose tissues are exposed to an excess of long-chain fatty acids, unless leptin action increases their oxidation sufficiently, unoxidized fatty acids enter nonoxidative pathways. While initially they are sequestered as harmless neutral fat, ultimately some will enter more toxic pathways. One of these, the de novo ceramide pathway, has been implicated in the lipoapoptosis of beta-cells and myocardiocytes of congenitally obese rats in which leptin action is defective. Here we review the mechanisms of lipoapoptosis and the diseases that result from this cause of a diminishing cell population of these organs. We suggest that some of the components of the metabolic syndrome of obese humans and the sarcopenia of aging may be result of failure of leptin liporegulation to prevent lipid overload of lean body mass and lipoapoptosis in certain organ systems.

Acyltransferases↗