910 resultados para LDL-Receptor Related Proteins
Resumo:
Mouse mammary tumor virus (MMTV) contained six major proteins, identified as gp55, gp33, p25, pp20, p12, and p10. Immunoprecipitation of cytoplasmic extracts from MMTV-infected, pulse-labeled cells identified three MMTV core-specific precursor proteins, termed Pr78('gag), Pr110('gag), Pr110('gag), and Pr180('gag+). The major intracellular core-specific precursor polyprotein, Pr78('gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, and p10. Pr110('gag) contained all but one of the leucine-containing tryptic peptides of Pr78('gag), plus several additional peptides. In addition to Pr78('gag) and Pr110('gag), monospecific antisera to virion p12 and p25 also precipitated from pulse-labeled cells a small amount of Pr180('gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78('gag) and Pr110('gag) plus several additional peptides. By analogy to type-C viral systems, Pr180('gag+) is presumed to represent a gag-pol-specific common precursor which is the major translation product in the synthesis of MMTV RNA-dependent-DNA polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two envelope-specific proteins, designated gPr76('env) and gP79('env). The major envelope-specific precursor, gPr76('env), could be labeled with radioactive glucosamine and contained antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A quantitatively minor glycoprotein, gP79('env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79('env) represents fucosylated gPr76('env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.^ A glycoprotein of 130,00 molecular weight (gP130) was precipitable from the cytoplasm of GR-strain mouse mammary tumor cells by a rabbit antiserum (anti-MMTV) to Gr-strain mouse mammary tumors virus (GR-MMTV). Two dimensional thin layer analysis of ('35)S-methionine-containing peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides were shared by gP130 and gPr76('env). Six of ten p25 peptides and four more core-related peptides were shared by Pr78('gag) and gP130. Protein gP130 also contained several tryptic peptides not found in gPr76('env), or in the core protein precursors Pr78('gag), Pr110('gag), or Pr180('gag+). both gP130 and a second protein, p30, were found in immunoprecipitates of detergent disrupted, isotopically labeled GR-MMTV treated with anti-MMTV serum. Results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences which are not related to viral structural proteins. These gP130-unique peptides are evidently host specific. Polyproteins consisting of juxtaposed host- and virus-related protein tracts have been implicated in the process of cell transformation in other mammalian systems. Therefore, gP130 may be instrinsic to the oncogenic potential of MMTV. ^
Resumo:
After infection with the digenetic trematode Echinostoma paraensei, hemolymph of the snail Biomphalaria glabrata contains lectins comprised of 65-kDa subunits that precipitate polypeptides secreted by E. paraensei intramolluscan larvae. Comparable activity is lacking in hemolymph of uninfected snails. Three different cDNAs with sequence similarities to peptides derived from the 65-kDa lectins were obtained and unexpectedly found to encode fibrinogen-related proteins (FREPs). These FREPs also contained regions with sequence similarity to Ig superfamily members. B. glabrata has at least five FREP genes, three of which are expressed at increased levels after infection. Elucidation of components of the defense system of B. glabrata is relevant because this snail is an intermediate host for Schistosoma mansoni, the most widely distributed causative agent of human schistosomiasis. These results are novel in suggesting a role for invertebrate FREPs in recognition of parasite-derived molecules and also provide a model for investigating the diversity of molecules functioning in nonself-recognition in an invertebrate.
Resumo:
Chronic exposure to cocaine leads to prominent, long-lasting changes in behavior that characterize a state of addiction. The striatum, including the nucleus accumbens and caudoputamen, is an important substrate for these actions. We previously have shown that long-lasting Fos-related proteins of 35–37 kDa are induced in the striatum by chronic cocaine administration. In the present study, the identity and functional role of these Fos-related proteins were examined using fosB mutant mice. The striatum of these mice completely lacked basal levels of the 35- to 37-kDa Fos-related proteins as well as their induction by chronic cocaine administration. This deficiency was associated with enhanced behavioral responses to cocaine: fosB mutant mice showed exaggerated locomotor activation in response to initial cocaine exposures as well as robust conditioned place preference to a lower dose of cocaine, compared with wild-type littermates. These results establish the long-lasting Fos-related proteins as products of the fosB gene (specifically ΔFosB isoforms) and suggest that transcriptional regulation by fosB gene products plays a critical role in cocaine-induced behavioral responses. This finding demonstrates that a Fos family member protein plays a functional role in behavioral responses to drugs of abuse and implicates fosB gene products as important determinants of cocaine abuse.
Resumo:
The adenovirus E1A oncoprotein renders primary cells sensitive to the induction of apoptosis by diverse stimuli, including many anticancer agents. E1A-expressing cells accumulate p53 protein, and p53 potentiates drug-induced apoptosis. To determine how E1A promotes chemosensitivity, a series of E1A mutants were introduced into primary human and mouse fibroblasts using high-titer recombinant retroviruses, allowing analysis of E1A in genetically normal cells outside the context of adenovirus infection. Mutations that disrupted apoptosis and chemosensitivity separated into two complementation groups, which correlated precisely with the ability of E1A to associate with either the p300/CBP or retinoblastoma protein families. Furthermore, E1A mutants incapable of binding RB, p107, and p130 conferred chemosensitivity to fibroblasts derived from RB-deficient mice, but not fibroblasts from mice lacking p107 or p130. Hence, inactivation of RB, but not p107 or p130, is required for chemosensitivity induced by E1A. Finally, the same E1A functions that promote drug-induced apoptosis also induce p53. Together, these data demonstrate that p53 accumulation and chemosensitivity are linked to E1A’s oncogenic potential, and identify a strategy to selectively induce apoptosis in RB-deficient tumor cells.
Resumo:
The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.
Resumo:
Quiescent mouse embryonic C3H/10T½ cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T½ cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T½ cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T½ cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of β-catenin, suggesting that SARPs interfere with the Wnt–frizzled proteins signaling pathway.
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Taking advantage of the ongoing Dictyostelium genome sequencing project, we have assembled >73 kb of genomic DNA in 15 contigs harbouring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analysed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residue C-terminal extension and RacA a 400 residue C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB.
Resumo:
p300 and CBP participate as transcriptional coregulators in the execution of a wide spectrum of cellular gene expression programs controlling cell differentiation, growth and homeostasis. Both proteins act together with sequence-specific transcription factors to modify chromatin structure of target genes via their intrinsic acetyltransferase activity directed towards core histones and some transcription factors. So far, p300-related proteins have been described in animals ranging from Drosophila and Caenorhabditis elegans to humans. In this report, we describe p300/CBP-like polypeptides in the plant Arabidopsis thaliana. Interestingly, homology between animal and plant p300/CBP is largely restricted to a C-terminal segment, about 600 amino acids in length, which encompasses acetyltransferase and E1A-binding domains. We have examined whether this conservation in sequence is paralleled by a conservation in function. The same amino acid residues critical for acetyltransferase activity in human p300 are also critical for the function of one of the plant orthologs. Remarkably, plant proteins bind to the adenovirus E1A protein in a manner recapitulating the binding specificity of mammalian p300/CBP. The striking conservation of an extended segment of p300/CBP suggests that it may constitute a functional entity fulfilling functions that may be essential for all metazoan organisms.
Resumo:
Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that are found in the HDA1 complex and show that HDA1 interacts with itself and with the HDA2-HDA3 subcomplex to form a likely tetramer. These interactions are necessary for catalytic activity because mutations in any of the three components disrupt activity both in vitro and in vivo. In this respect the HDA1 complex differs from yeast RPD3, which has components such as SIN3 that are not essential for activity in vitro, and yeast HOS3, which has intrinsic in vitro activity as a homodimer in the absence of other subunits.
Resumo:
Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.
Resumo:
The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP–CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.
Resumo:
The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.
Resumo:
The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.
Resumo:
To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.