Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevis model of retinal degenerative disease.

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis.

Huntington's disease is characterised by the death of cortical and striatal neurons, and is the result of an expanded polyglutamine tract in the Huntingtin protein [1]. Huntingtin is present on both endocytic and secretory membrane organelles but its function is unclear [2,3]. Rab GTPases regulate both of these transport pathways [4]. We have previously shown that Rab8 controls polarised membrane transport by modulating cell morphogenesis [5]. To understand Rab8-mediated processes, we searched for Rab8-interacting proteins by the yeast two-hybrid system. Here, we report that Huntingtin is linked to the Rab8 protein through FIP-2, a tumour necrosis factor-alpha (TNF-alpha)-inducible coiled-coil protein related to the NEMO protein [6,7]. The activated form of Rab8 interacted with the amino-terminal region of FIP-2, whereas dominant-negative Rab8 did not. Huntingtin bound to the carboxy-terminal region of FIP-2. Coexpressed FIP-2 and Huntingtin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures, and FIP-2 promoted cell polarisation in a similar way to Rab8. We propose a model in which Huntingtin, together with FIP-2 and Rab8, are part of a protein network that regulates membrane trafficking and cellular morphogenesis.

Immunoprecipitation of steroidogenic enzyme autoantigens with autoimmune polyglandular syndrome type I (APS I) sera; further evidence for independent humoral immunity to P450c17 and P450c21.

Three steroidogenic P450 cytochromes, steroid 17alpha-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc), have been described as autoantigens in APS I. In this study we report an immunoprecipitation assay for the detection of autoantibodies to these three enzymes using in vitro 35S-labelled antigens. Overall, 33 out of 46 (72%) patients with APS I had autoantibodies to at least one of the three proteins and each protein was recognized by patient sera with equal frequency. A higher rate of autoantibody positivity was observed in APS I patients with Addison's disease compared with patients without Addison's disease (85% versus 39%). All 11 patients with ovarian failure had anti-P450c17 or anti-P450scc antibodies. The immunoprecipitation results with P450c17, P450c21 and P450scc correlated well with the results obtained by immunoblotting assays. In addition, the steroidogenic enzymes 11beta-hydroxylase (P450c11beta), aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase (3betaHSD) and adrenodoxin were studied by immunoprecipitation assay, but no reaction was found either with 46 APS I or with 26 healthy control sera. To study the suggested immunological cross-reactivity between P450c17 and P450c21 enzymes, nine APS I patient sera were preabsorbed with bacterially expressed P450c17 or P450c21 and subsequently used in immunoprecipitation assay. The absorption experiments clearly indicated that the preincubation inhibited only the reactivity of corresponding antigen, suggesting independent autoantibody response to the two enzymes. Our results suggest that the immune response to some but not to all steroidogenic enzymes is a specific feature of APS I that may be pathogenically significant.

Expression and intracellular localization of catechol O-methyltransferase in transfected mammalian cells.

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

VIP17/MAL, a proteolipid in apical transport vesicles.

VIP17 is a proteolipid enriched in the CHAPS-insoluble complexes from MDCK cells, and a candidate component of the molecular machinery responsible for the sorting and targeting of proteins to the apical surface. Cloning and sequencing of the cDNA encoding the protein revealed that it is the canine homolog of the human and rat MAL proteins. Analysis by immunofluorescence microscopy of epitope-tagged VIP17/MAL expressed transiently in BHK cells and stably in MDCK cells revealed a perinuclear, vesicular, and plasmalemmal staining. In MDCK cells the distribution was mainly in vesicular structures in the apical cytoplasm. These and other results suggest that VIP17/MAL is an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane.

N-glycans as apical sorting signals in epithelial cells.

In epithelial Madin-Darby canine kidney (MDCK) cells newly synthesized molecules are sorted in the trans-Golgi network and directly delivered to their apical and basolateral surface destinations. Sorting is mediated by signals in the cytoplasmic domains of basolateral transmembrane proteins whereas glycosylphosphatidylinositol-linked proteins have apical sorting information in their glycolipid tails. Signals for apical transmembrane proteins are thought to reside in their ectodomains, because truncated forms lacking the cytoplasmic tail and the membrane anchor are secreted apically. Here we demonstrate that carbohydrates act as an apical targeting signal for secretory proteins. Growth hormone, which is non-glycosylated and secreted from both sides of MDCK cell layers, is secreted from the apical side when glycosylated. Thus glycans not only play a general role in protein folding but also appear to function in protein sorting in biosynthetic traffic.

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

The alphavirus replicase protein nsP1 is membrane-associated and has affinity to endocytic organelles.

In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles. In this paper, we show that when nsP1 was expressed alone in HeLa cells with the aid of the recombinant T7 RNA polymerase vaccinia (vTF7-3) virus system, it was tightly associated with intracellular smooth membranes. The membrane association may be due to acylation, since nsP1 could be labeled with [3H]palmitic acid in both Semliki Forest virus-infected and nsP1-transfected HeLa cells. Release of the 3H-label by alkaline methanolysis suggests that the palmitate was associated with nsP1 via an ester bond. Pulse-chase experiments done on nsP1-transfected cells revealed that this protein was rapidly associated with the membranes. After synchronizing the synthesis of the nsP1 gene product in transfected cells, nsP1 appeared first at the plasma membrane and thereafter on vesicles, many of which contained the endosomal transferrin receptor marker. Later, nsP1 appeared on large vacuoles, which contained the lysosome specific h-lamp-1 protein. Membrane association of nsP1, and its affinity to endosomes and lysosomes, suggest a role of this protein in the biogenesis of the alphavirus-specific RNA replication complex.

Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli.

We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsP1 both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPV-nsP1. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsP1 were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsP1-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsP1 were in aggregates. nsP1 expressed in E. coli at 37 degrees C sedimented at 15,000 x g, whereas after expression at 15 degrees C, both nsP1 and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the "soluble" nsP1-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsP1-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [3H]methyl group from S-adenosylmethionine to GTP for a 60-min period. The enzymes from the three sources had similar substrate specificities and Km values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m7GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsP1 methyltransferase are discussed.

ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2.

The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein. One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein. On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities. Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system. Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography. The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA. The activities were not found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide. Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity. We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn). No nucleoside triphosphatase activity was associated with this mutant protein.

Nuclear targeting of Semliki Forest virus nsP2.

The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.

A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies.

We describe a simple protocol that allows the retrieval of masked or hidden intracellular antigens in cultured cells. The protocol is based on the exposure of paraformaldehyde-fixed and Triton X-100-permeabilized cells to guanidine hydrochloride (GdnHCl). We used it for localization of different antigens in BHK-21 cells by immunofluorescence microscopy. Our results showed that five out of six antibodies, initially unreactive, became excellent localization tools when used in conjunction with GdnHCl. Denaturation of fixed cells with GdnHCl did not affect the overall cell architecture, when monitored with different organelle-specific antibodies. In two cases out of 17 the antigenic sites were lost after denaturation. However, this problem could be partially overcome by including low amounts of glutaraldehyde in the fixative. We think that this method could be generally useful in immunofluorescence localization studies, particularly in cases where the antibodies are known to react only with denatured antigens.

Rapid affinity-purification and biotinylation of antibodies.

A simple and rapid method to affinity-purify and biotinylate antibodies was developed. The method utilizes separation of antigens by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose and binding of the antibodies to the specific antigen. The antibodies are biotinylated, while still bound to the antigen, thus avoiding the conjugation of the active antigen-binding sites of the antibodies. These antibodies have been successfully used in double-label immunofluorescence studies, but they should be likewise applicable in other immunological protocols.

Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2.

Semliki Forest virus nonstructural protein nsP2, when expressed in the absence of other viral proteins, is transported into the nucleus, with a specific enrichment in nucleoli. This implies that information for its nuclear targeting must be in nsP2 itself. To define the nuclear and nucleolar targeting signals of nsP2, a series of in-frame deletions were made in its coding sequence. We have identified one amino-terminal (residues 25-110) and another carboxy-terminal (residues 634-661) deletion, both of which rendered nsP2 cytoplasmic. The carboxy-terminal region had a pentapeptide sequence P 648R RRV, which resembles some of the known nuclear localization signals. When the three arginines were substituted with aspartic acids, the mutant nsP2 was localized in the cytoplasm. Especially arginines 648-649 were shown to be critical. The carboxy-terminal 232 amino acid residues of nsP2 were able to translocate beta-galactosidase to the nucleus as a fusion protein, whereas the amino-terminal 110 residues failed to do so. Studies with different beta-galactosidase-nsP2 fusions showed that the pentapeptide is necessary for nuclear transport but that its activity is strongly affected by the context. The deletion analysis indicated also that the nuclear localization signal was not sufficient for nucleolar targeting of nsP2. The nucleolar targeting sequence of nsP2 was tentatively mapped between residues 470 and 539, and at least residues 470-489 are an essential part of it.

Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme.

To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.