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At least 559 records · Page 31Linked to original sources
PubMed2006

Distinct subcellular localization for constitutive and agonist-modulated palmitoylation of the human delta opioid receptor.

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human delta opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (Mr 55,000), [3H]palmitate incorporation into the receptor precursor (Mr 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonist-mediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.

Cell Line↗
PubMed2000

Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

Antigens, Differentiation, Myelomonocytic↗
PubMed1994

Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.

The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.

Actins↗
PubMed2000

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis.

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

Amino Acid Sequence↗
PubMed2004

Trafficking of Lyn through the Golgi caveolin involves the charged residues on alphaE and alphaI helices in the kinase domain.

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced "closed conformation" but not by kinase inactivation. Four residues (Asp346 and Glu353 on alphaE helix, and Asp498 and Asp499 on alphaI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an "open conformation," are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.

Amino Acid Sequence↗
PubMed2006

Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase.

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

Acylation↗
PubMed2002

DOLOP--database of bacterial lipoproteins.

UNLABELLED: Bacterial lipoproteins and lipid modification are gaining importance owing to their essential nature, roles in pathogenesis and interesting commercial applications. We have created an exclusive knowledge base for bacterial lipoproteins by processing information from 510 entries to provide a list of 199 distinct lipoproteins with relevant links to molecular details. Features include functional classification, predictive algorithm for query sequences, primary sequence analysis and lists of predicted lipoproteins from 43 completed bacterial genomes along with interactive information exchange facility. AVAILABILITY: The website called Database Of bacterial LipOProteins (DOLOP) is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop along with additional information on the biosynthetic pathway, supplementary material and other related figures.

Algorithms↗
PubMed2006

CSS-Palm: palmitoylation site prediction with a clustering and scoring strategy (CSS).

UNLABELLED: Palmitoylation is an important post-translational lipid modification of proteins. Unlike prenylation and myristoylation, palmitoylation is a reversible covalent modification, allowing for dynamic regulation of multiple complex cellular systems. However, in vivo or in vitro identification of palmitoylation sites is usually time-consuming and labor-intensive. So in silico predictions could help to narrow down the possible palmitoylation sites, which can be used to guide further experimental design. Previous studies suggested that there is no unique canonical motif for palmitoylation sites, so we hypothesize that the bona fide pattern might be compromised by heterogeneity of multiple structural determinants with different features. Based on this hypothesis, we partition the known palmitoylation sites into three clusters and score the similarity between the query peptide and the training ones based on BLOSUM62 matrix. We have implemented a computer program for palmitoylation site prediction, Clustering and Scoring Strategy for Palmitoylation Sites Prediction (CSS-Palm) system, and found that the program's prediction performance is encouraging with highly positive Jack-Knife validation results (sensitivity 82.16% and specificity 83.17% for cut-off score 2.6). Our analyses indicate that CSS-Palm could provide a powerful and effective tool to studies of palmitoylation sites. AVAILABILITY: CSS-Palm is implemented in PHP/PERL+MySQL and can be freely accessed at http://bioinformatics.lcd-ustc.org/css_palm/ CONTACT: yaoxb@ustc.edu.cn; xuyn@bmb.uga.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bionformatics online.

Algorithms↗
PubMed2003

Rat mammary carcinogenesis induced by in situ expression of constitutive Raf kinase activity is prevented by tethering Raf to the plasma membrane.

Mammary carcinogenesis induced through expression of activated Raf was investigated using a model in which retroviral vectors were infused into the central ducts of rat mammary glands. This model allows efficient expression of experimental proteins in a small fraction of endogenous mammary epithelial cells in situ. We previously reported that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands. We show here that mammary gland carcinogenesis is rapidly induced by the expression of c-Raf-1 kinase that is activated by N-terminal truncation (Delta-Raf). Interestingly, targeting Raf to the plasma membrane via C-terminal fusion with Ras membrane localization signals (Raf-Caax) induces Raf kinase activity that transforms 3T3 cells more frequently than Delta-Raf, yet in situ expression of Raf-Caax does not induce mammary carcinomas. To investigate these contrasting results and begin elucidating the mechanisms of Raf-induced mammary carcinogenesis, we combined both activating mutations (N-terminal truncation and C-terminal membrane localization motifs) in one Raf construct (Delta-Raf-Caax). While Delta-Raf-Caax transforms 3T3 cells more efficiently than Delta-Raf or Raf-Caax, in situ expression of Delta-Raf-Caax does not induce carcinomas in vivo, demonstrating that lipid modification on the C-terminus of Delta-Raf negates its oncogenic potential in rat mammary gland.

3T3 Cells↗
PubMed2003

Prenylation inhibitors: a novel class of antiviral agents.

Prenylation is a site-specific lipid modification of proteins. Although first described for a variety of cellular proteins, it has become apparent that viruses can also make use of this post-translational modification provided by their host cells. Depriving a virus access to prenylation can have dramatic effects on the targeted virus's life cycle. Selective pharmacological inhibitors of prenylating enzymes have been developed and shown to have potent antiviral effects in both in vitro and in vivo systems. Because prenylation inhibitors target a host cell function, are available in oral form and are surprisingly well tolerated in human trials, these compounds represent an attractive new class of antiviral agents with potential for broad-spectrum activity. After a brief outline of host cell prenylation pathways, we review below the development of prenylation inhibition as an antiviral strategy applied to a prototype target, hepatitis delta virus (HDV), and discuss the potential application of prenylation inhibitors to a broad range of other viruses.

Antiviral Agents↗
PubMed2005

Citrate anticoagulation abolishes degranulation of polymorphonuclear cells and platelets and reduces oxidative stress during haemodialysis.

BACKGROUND: During haemodialysis (HD), polymorphonuclear cells (PMNs) and platelets are activated and release various granule products, including myeloperoxidase (MPO) and platelet factor 4 (PF4). MPO triggers the generation of reactive oxygen species, leading to irreversible protein, carbohydrate and lipid modification. PF4 probably also contributes to oxidative stress. As previously shown, HD-induced PMN degranulation is almost completely abolished during citrate anticoagulation, most probably due to its calcium chelation ability. METHODS: In the present study, apart from HD-induced PMN and platelet degranulation, oxidative stress was analysed during three modes of anticoagulation. Heparin, dalteparin and citrate (HDhep, HDdal and HDcit) were compared in a randomized, crossover fashion in eight chronic HD patients. Multiple blood samples were taken during the third HD session of each modality, from both the afferent and efferent line. Besides the degranulation markers MPO and PF4, various markers of oxidative stress were measured, including oxidized low-density lipoprotein (ox-LDL), malondialdehyde (MDA) and carboxymethyllysine (CML). RESULTS: During HDhep and HDdal, marked degranulation was observed shortly after the start of HD. In contrast, during HDcit, PF4 and MPO levels remained unaltered, suggesting no release at all. After 1 week of HDcit, ox-LDL levels were markedly reduced [median 26% (3-65%), P=0.01], if compared with HDhep and HDdal. As regards MDA and CML, no differences were found. CONCLUSIONS: This study shows first, that HD-induced PMN and platelet degranulation are early, most probably calcium-dependent processes and, secondly, that the formation of ox-LDL is clearly dependent on the type of anticoagulant applied.

Adult↗
PubMed1998

Mutagenesis of putative acylation sites alters function, localization, and accumulation of a Gi alpha subunit of the chestnut blight fungus Cryphonectria parasitica.

Targeted disruption of cpg-1, a gene encoding the G protein Gi alpha subunit, CPG-1, in the chestnut blight fungus, Cryphonectria parasitica, results in reduced mycelial growth, reduced orange pigmentation, loss of virulence, loss of asexual sporulation, and female infertility. We report the development of a complementation system for cpg-1 null mutants and its use to evaluate the in vivo consequences of mutating conserved putative CPG-1 myristoylation (G2) and palmitoylation (C3) sites. Independent mutations of the two putative acylation sites differentially altered complex fungal biological processes, including virulence, and modified CPG-1 membrane association. Results of combined Northern (RNA) and Western (immunoblot) analysis also indicated a role for lipid modification in post-transcriptional regulation of CPG-1 accumulation.

Acylation↗
PubMed2005

Palmitoylation at Cys574 is essential for MT1-MMP to promote cell migration.

MT1-MMP is a type I transmembrane proteinase that promotes cell migration and invasion. Here, we report that MT1-MMP is palmitoylated at Cys574 in the cytoplasmic domain, and this lipid modification is critical for its promotion of cell migration and clathrin-mediated internalization. The palmitoylation-defective mutant (C574A) failed to promote cell migration and was not internalized through clathrin pathway like wild-type, but it was internalized through the caveolae pathway. Reintroducing a cysteine at different positions in the cytoplasmic tail of the C574A mutant revealed that the position of the palmitoylated cysteine relative to LLY573, a motif that interacts with mu2 subunit of adaptor protein 2, is critical for the cell motility-promoting activity of MT1-MMP and its clathrin-mediated internalization. Taken together, palmitoylation of MT1-MMP is one of the key posttranslational modifications that determines MT1-MMP-dependent cell migration.

Amino Acid Motifs↗
PubMed2000

Perillyl alcohol, an inhibitor of geranylgeranyl transferase, induces apoptosis of immortalized human vascular smooth muscle cells in vitro.

The isoprenoids geranylgeraniol and farnesol are required for lipid modification of several important cellular proteins. In this study the effect of perillyl alcohol (PA), an inhibitor of geranylgeranyl transferase, was examined on proliferation and apoptosis of immortalized human vascular smooth muscle cells (HVSMCs) derived from saphenous vein. PA induced a time- and concentration-dependent inhibition of cell proliferation over 3 days and 48 h exposure to PA inhibited [methyl-3H-thymidine incorporation induced by 10% fetal calf serum in a concentration-dependent manner. Flow cytometry analysis indicated that PA reduced the proportion of cells in S phase and increased apoptosis. PA-induced apoptosis was confirmed by terminal deoxynucleotidyl transferase (TdT) reagent-based immunocytochemistry. In contrast, the selective inhibitors of farnesyl transferase B581 and BZA-5B were without effect. In view of the proposed role of proliferation and apoptosis in myointimal hyperplasia, inhibitors of geranylgeranyl transferases may have therapeutic potential in cardiovascular disease.

Alkyl and Aryl Transferases↗
PubMed1996

Unravelling high density lipoprotein-apolipoprotein metabolism in human mutants and animal models.

Apolipoprotein A-I plays an essential structural and functional role in HDL metabolism and apolipoprotein A-II has important effects on HDL metabolism and function. Kinetic studies in humans have established that variation in plasma HDL-cholesterol and apolipoprotein A-I concentrations is primarily determined by variation in the rate of apolipoprotein A-I catabolism. In contrast, plasma apolipoprotein A-II levels are primarily determined by the rate of apolipoprotein A-II production. Genetic factors play an important role in modulating the plasma levels of HDL-cholesterol and apolipoproteins A-I and A-II. Studies in humans have established that mutations in genes encoding enzymes that esterify cholesterol (lecithin : cholesterol acyltransferase), transfer cholesterol (cholesteryl ester transfer protein) and hydrolyze lipids (hepatic lipase, lipoprotein lipase) regulate HDL-cholesterol and apolipoprotein A-I levels by modifying the lipid content (and therefore the size) of HDL particles. Recent studies in transgenic and knockout animals have confirmed the key role of HDL lipid-modifying proteins in HDL, apolipoprotein A-I and apolipoprotein A-II metabolism and have expanded our understanding of the role of lipid modification in determining plasma concentrations of HDL-cholesterol and apolipoprotein A-I, as well as the potential functional roles of apolipoprotein A-II.

Animals↗
PubMedDate not supplied

Strategies for the prevention of atherosclerotic progression in women.

Atherosclerosis is an inflammatory and fibrotic process that begins early in life in females. The pathogenesis involves the oxidation of the low-density lipoprotein molecule aggravated by smoking, hypertension, lipid abnormalities, and hormonal changes. Risks for progression of atherosclerosis can now be tabulated for the female based on age, cholesterol, smoking, high-density lipoprotein, and systolic blood pressure. During the reproductive years, emphasis should be placed on lifestyle changes, but women at increased risk for diabetes should be aggressively treated with lipid-lowering agents. During the menopausal phase of life, an important consideration is the use of hormone replacement along with lifestyle changes, smoking cessation, blood pressure control, and lipid modification. In the female with established coronary heart disease, standard cardiac medications are indicated along with more aggressive approaches to risk factors and target goals for cholesterol, low-density lipoprotein, and triglycerides. The effect of hormone replacement in established coronary artery disease is uncertain. Therefore, strategies for slowing the progression of atherosclerosis should begin during the reproductive years, with particular emphasis in patients during menopause and in patients with coronary heart disease.

Arteriosclerosis↗
PubMed2002

Many carried meningococci lack the genes required for capsule synthesis and transport.

Of 830 Neisseria meningitidis isolates obtained from healthy carriers in Bavaria, Germany, 136 (16.4%) lacked the operons necessary for the synthesis, lipid modification, and transport of capsular polysaccharide. These operons were replaced by a non-coding intergenic region either 113 or 114 bp in length, termed here the capsule null locus (cnl). Comparisons of the nucleotide sequence of this region in the meningococcus and its acapsulate relatives, Neisseria gonorrhoeae and Neisseria lactamica, revealed six distinct sequence variants (cnl-1 to cnl-6), with a total of 10 nucleotide substitutions and three indels. With the exception of one 4 bp insertion, which was unique to a gonococcal isolate, all of the individual sequence changes were present in the N. lactamica isolates examined. The meningococcal isolates with a cnl belonged to one of four otherwise genetically diverse genetic groupings: the ST-53 and ST-1117 complexes (75 isolates); the ST-845 complex (12 isolates); the ST-198 and 1136 complexes (46 isolates), and the ST-44 complex (one isolate). These data demonstrated that a substantial proportion of carried meningococci were incapable of capsule production, that the cnl circulated within Neisseria populations by horizontal genetic exchange, and that the expression of a polysaccharide capsule was not a requirement for person-to-person transmission of certain meningococcal lineages.

Bacterial Capsules↗