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Biomedical subjects

E Ikonen

Publications and source records attributed to E Ikonen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Family of human oxysterol binding protein (OSBP) homologues. A novel member implicated in brain sterol metabolism.

Oxysterol binding protein (OSBP) is a cytosolic protein that undergoes ligand-induced binding to the Golgi apparatus and has been implicated in the regulation of cellular cholesterol metabolism. In the yeast Saccharomyces cerevisiae an OSBP homologue is involved in membrane trafficking through the Golgi complex. Prompted by the multitude of OSBP-related genes in the yeast genome, we carried out a search for human expressed sequence tags (ESTs) displaying homology to the sterol-binding domain of OSBP. This revealed a minimum of six novel OSBP-related proteins, designated ORP-1 to ORP-6. ORP cDNA probes were generated by reverse transcription-PCR from human liver mRNA, and used for Northern blot analysis of human tissue transcript panels. This verified that each of them represents a different gene product and showed that they display distinct tissue-specific expression patterns. The ORP-1 and -2 mRNA expression levels were similar to or higher than that of OSBP while the ORP-3 to -6 mRNAs were detected at lower levels in specific tissues. The most abundantly expressed new gene, ORP-1, was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line. This indicates that ORP-1 may play an important role in maintaining the sterol balance in cells of the central nervous system. Together with OSBP, the identified gene products constitute a novel human protein family that may provide a link between organellar sterol status and membrane dynamics.

Binding Sites↗
PubMed1998

Caveolin-1 and -2 in the exocytic pathway of MDCK cells.

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.

Amino Acid Sequence↗
PubMed1998

Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells.

The targeting of proteins and lipids to the cell surface domains of polarized cells is not a simple bulk flow process but requires sorting into distinct apical and basolateral pathways from the trans-Golgi network. Here, we describe the sorting determinants in the cargo molecules, the cellular sorting machineries responsible for the hierarchical read-out of the signals, and the mechanisms of cargo delivery. Furthermore, we discuss the implications of these findings for protein-lipid interactions in other cellular machineries.

Animals↗
PubMed1997

Functional rafts in cell membranes.

A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.

Animals↗
PubMed1997

Intracellular and secreted Alzheimer beta-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons.

Cerebral plaques containing beta-amyloid (beta A4) represent an invariant pathological feature of Alzheimer disease (AD). beta A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In non-neuronal cells beta A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of beta A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of beta A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular beta A4. We also show that a common feature of two types of APP mutations ("Swedish" and "London") implicated in early-onset AD is their increased production of C-terminally elongated beta A4 (beta 42), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular beta A4 and also the main victims in AD, these findings may provide an important link between beta A4 and neurodegeneration.

Amyloid beta-Peptides↗
PubMed1997

Molecular mechanisms of intracellular cholesterol transport.

Several examples in recent literature show that the molecular principles governing intracellular cholesterol transport are starting to emerge. Two previously cloned proteins were discovered to play a role in sterol transport of steroidogenic cells: the scavenger receptor BI as an HDL receptor and steroidogenic acute regulatory protein in the transport of cholesterol to mitochondria. Increasing evidence suggests the involvement of multidrug resistance proteins in sterol trafficking from the cell surface to the endoplasmic reticulum or across the plasma membrane. Specialized plasma membrane invaginations, caveolae, were implicated in cholesterol efflux, and the abundant caveolar protein, caveolin-1 which belongs to a newly discovered caveolin family of proteins, was shown to be a cholesterol-binding membrane protein.

Biological Transport↗
PubMed1997

Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles.

A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.

Actins↗
PubMed1996

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.

Alzheimer Disease↗
PubMed1996

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.

Animals↗
PubMed1995

Preliminary phenotypic map of chromosome 4p16 based on 4p deletions.

We have collected and analyzed clinical information from 11 patients with chromosome 4p deletions or rearrangements characterized by various molecular techniques. Comparing the extent of these patients' deletions with their respective clinical presentations led to the proposal of a preliminary phenotypic map of chromosome 4p. This map consists of regions which, when deleted, are associated with specific clinical manifestations. Nonspecific changes such as mental and growth retardation are not localized, and probably result from the deletion of more than one gene or region. The region associated with most of the facial traits considered typical in Wolf-Hirschhorn syndrome (WHS) patients coincides with the currently proposed WHS critical region (WHSCR), but some anomalies commonly seen in WHS appear to map outside of the WHSCR. The observation of clinodactyly in 2 patients with nonoverlapping deletions allows assignment of these defects to at least 2 separate regions in 4p16. These initial observations and attempts at genotype/phenotype correlation lay the groundwork for identifying the genetic basis of these malformations, a common objective of gene mapping efforts and chromosome deletion studies.

Abnormalities, Multiple↗
PubMed1995

Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells.

We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.

Animals↗
PubMed1995

Intracellular routing of human amyloid protein precursor: axonal delivery followed by transport to the dendrites.

A characteristic neuropathological feature of Alzheimer's disease is the cerebral deposition of amyloid plaques. These deposits contain beta A4 amyloid peptide, a cleavage product of the transmembrane protein amyloid protein precursor (APP). Despite numerous studies on the processing of the different APP isoforms in non-neuronal cells, little is known about its sorting and transport in neurons of the central nervous system (CNS). To analyze this question we expressed in cultured rat hippocampal neurons the human APP 695, tagged at its N-terminus with the myc epitope, using the Semliki forest virus (SFV) expression system. APP was first delivered from the cell body to the axon and later appeared also in the dendrites. Inhibition of protein synthesis at the time of axonal expression did not block the late appearance of the protein in the dendrites. An antibody directed against the myc tag, bound to the cell surface at 4 degrees C at the time of axonal APP expression, could be chased to the dendritic domain after subsequent incubation at 37 degrees C. These results suggest that the newly synthesized APP, after initial axonal delivery, may be transported to the dendrites by a transcytotic mechanism. The routing of APP in polarized neurons is different from that of polarized epithelial cells, in which the protein is delivered basolaterally, arguing for neuronal specific sorting and processing mechanisms.

Amino Acid Sequence↗
PubMed1995

Prohibitin, an antiproliferative protein, is localized to mitochondria.

Prohibitin is a ubiquitously expressed protein with antiproliferative properties. When rat prohibitin tagged with a carboxy-terminal c-Myc epitope was expressed in baby hamster kidney cells the protein was targeted to mitochondria. In immunofluorescence microscopy prohibitin colocalized with a mitochondrial marker E3. Immunoelectron microscopy revealed that prohibitin was associated with the periphery of mitochondria. The amino-terminus of prohibitin shares characteristics of the known mitochondrial import signals, and positioning of the tag at the N-terminus causes accumulation of the protein in the cytoplasm. These findings help to direct functional studies on prohibitin and suggest that a mitochondrial protein may act as a tumor suppressor.

Amino Acid Sequence↗
PubMed1995

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.

Animals↗
PubMed1995

Intracellular sorting of aspartylglucosaminidase: the role of N-linked oligosaccharides and evidence of Man-6-P-independent lysosomal targeting.

Aspartylglucosaminidase (AGA, E.C. 3.5.1.26) is a soluble lysosomal hydrolase that participates in the degradation of glycoproteins. Here we analyzed the special features in the intracellular targeting of this dimeric amidohydrolase, especially the role of N-linked sugars and their phosphorylation in transport and activity of heterodimeric aspartylglucosaminidase, using in vitro mutagenesis and transient expression of mutant polypeptides in COS cells. The single N-glycosylation sites of both the alpha and beta subunits were destroyed individually and in combination. Just one remaining N-glycosylation site on either subunit was sufficient for normal processing into subunits and lysosomal transport, but the totally nonglycosylated enzyme, although active and processed into subunits, was not transported into lysosomes and became trapped in the endoplasmic reticulum (ER) or secreted. The intracellular targeting of AGA was partially disturbed by the lack of glycosylation in the beta subunit, resulting in accumulation of dimeric, active polypeptides in the ER, whereas lack of oligosaccharides in the alpha subunit did not affect the intracellular targeting of AGA. N-glycans in the beta subunit were found to be essential for the long-term stability of the polypeptide in the cell, but not for initial folding or subunit processing into the active dimeric molecule. Both subunits have two glycosylation isoforms. Both forms of the alpha subunit were found to be phosphorylated, whereas only one of the two glycosylation isoforms of the beta subunit is phosphorylated. The mutant enzyme with nonglycosylated alpha subunit and nonphosphorylated beta subunit is transported into lysosomes, suggesting that AGA is capable of using an alternative, mannose-6-phosphate receptor-independent routing into lysosomes.

Aspartylglucosylaminase↗