Academic literature on the topic 'Golgo 13 II'

Pulmonary surfactant consists of phospholipids and proteins that form a stable monolayer at the surface of the alveoli to prevent lung collapse. Surfactant protein C (SP-C) is a hydrophobic 4-kDa palmitoylated protein derived from a 21-kDa precursor. We determined the membrane insertion, proteolytic processing, and subcellular location of 21-kDa proSP-C. In vitro, proSP-C associated with canine microsomes, and the NH2-terminal of proSP-C was protected from digestion with proteinase K, suggesting that proSP-C was inserted in a type III transmembrane configuration. Treatment of freshly isolated rat type II cells with cerulenin blocked acylation of the 21-kDa precursor. Pulse-chase labeling of type II cells demonstrated proSP-C processing intermediates of 19, 16, and 13 kDa that contained the NH2-terminal of proSP-C. Proteolytic processing of proSP-C was inhibited by incubation at 20 degrees C, suggesting that processing of proSP-C begins in a late Golgi or post-Golgi compartment. Immunogold labeling of rat lung with an antiserum to the NH2-terminal of proSP-C identified proSP-C in the trans-Golgi and multivesicular bodies but not in lamellar bodies. These findings suggest that proSP-C processing takes place in the trans-Golgi and multivesicular bodies before SP-C is incorporated into lamellar bodies.

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.

Coat protein II (COPII)–mediated export from the endoplasmic reticulum (ER) involves sequential recruitment of COPII complex components, including the Sar1 GTPase, the Sec23/Sec24 subcomplex, and the Sec13/Sec31 subcomplex. p125A was originally identified as a Sec23A-interacting protein. Here we demonstrate that p125A also interacts with the C-terminal region of Sec31A. The Sec31A-interacting domain of p125A is between residues 260–600, and is therefore a distinct domain from that required for interaction with Sec23A. Gel filtration and immunodepletion studies suggest that the majority of cytosolic p125A exists as a ternary complex with the Sec13/Sec31A subcomplex, suggesting that Sec 13, Sec31A, and p125A exist in the cytosol primarily as preassembled Sec13/Sec31A/p125A heterohexamers. Golgi morphology and protein export from the ER were affected in p125A-silenced cells. Our results suggest that p125A is part of the Sec13/Sec31A subcomplex and facilitates ER export in mammalian cells.

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

Abstract Abstract 4028 Poster Board III-964 CDAII, the most frequent type of congenital dyserythropoietic anemia family, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblasts' morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Recent studies indicated that CDAII is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. Linkage analysis located the CDAII gene in a 5 cM region on chromosome 20 and several candidate genes have been excluded. We recently investigated the cytoplasmic proteome of human red blood cells (RBCs) using a combinatorial peptide ligand library as a capturing agent to amplify the signal of low- and very-low abundance proteins: 1578 proteins, most of them unexpected, were identified allowing a deep exploration of the RBC pathways (Roux-Dalvai, Mol Cell Proteomics, 2008). In this study we used a proteomic-genomic approach to identify the candidate gene for CDAII by matching the data on the cytoplasmic proteome of human RBC with the chromosomic localization of CDAN2 locus. The analysis of RBCs cytoplasmic proteome allowed us to identify 17 proteins codified by genes located in the chromosomic region between 20p11.23 and 20q11.23: SNX5, SEC23B, DTD1, NAT5, GINS1, BCL2L1, MAPRE1, CHMP4B, EIF2S2, AHCY, ACSS2, GSS, EIF6, CPNE1, EPB41L1, RPRD1B(C20orf77), TGM2. Most of them were excluded because found to be associated to other diseases, already excluded in CDAII by previous works, or because not related by function to CDAII. Among the remaining proteins we focus on SEC23B for its possible role in the endoplasmic reticulum-to Golgi trafficking and its localisation on 20p11, the region with the highest LOD-score in CDAII after the recent mapping of the markers on the current contigs (Denecke et al, Biochim Biophys Acta, 2009). The 20 exons and intronic flanking regions of SEC23B gene were analysed by direct sequencing in 16 CDA II patients from 13 families; 13 different mutations were detected among the 28 mutated alleles identified: 6 of them were missense, 2 frameshift, 1 splicing and 4 stop codon. All the missense mutations affected highly conserved aminoacids, and were not found in 100 normal alleles examined. Two of them (c.40C>T and c.325G>A) were detected in various unrelated patients. Despite all exons and flanking intronic regions were sequenced, one patient failed to show mutations and two cases displayed only one mutation, suggesting the possibility that a second gene could be involved in CDAII. Patients' data are summarised on the table (*,° = members of the same family). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles. Very recently it has been shown that knockdown of Zebrafish SEC23B leads to aberrant erythrocyte development (Schwarz et al, Nat Genet, 2009). Even if the exact function of human SEC23B is not completely clarified, abnormalities in this gene are likely to disturb ER-to Golgi trafficking affecting different glycosylation pathways, and ultimately accounting for the cellular phenotype observed in CDA II. Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 1 F N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 2 F C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 3 F Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 4 M Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 5 M S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 6 F C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 7 M C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 8° F N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 9° M N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 10° F N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 11 F N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 12* M S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 13* F S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 14 F N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 15 M N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 16 F S Italy 10.7 87 Yes ?/? ?/? ?/? Disclosures: No relevant conflicts of interest to declare.

AbstractProtein Z is a vitamin K–dependent glycoprotein that plays a role in the regulation of coagulation. A nucleotide substitution of G by C in exon II of the protein Z gene, resulting in the replacement of Glu-30 with Gln (E30Q), and a G to A transition at the 79th nucleotide in intron F (IntF79G/A) were heterozygously identified in a patient with a severe thrombotic tendency, whose plasma protein Z level was about 15% of normal. Other vitamin K–dependent coagulation factors were within normal ranges. Glu-30 is one of 13 γ-carboxylation sites in protein Z and is well conserved among vitamin K–dependent proteins. Expression studies revealed that the E30Q mutant was not released from synthesizing cells, although wild-type protein Z was readily secreted in a vitamin K–dependent fashion. The E30Q mutant was N-glycosylated, γ-carboxylated, and translocated from the endoplasmic reticulum (ER) to the Golgi in the presence of vitamin K, as was the wild type. Coexpression of E30Q with wild-type protein Z interfered with the secretion of the wild type, while only a minor or no effect was observed on the secretion of factor X and plasminogen. The IntF79A allele has been reported to be also associated with lowered protein Z levels.

A panel of mutant class II genes have been constructed using site-directed mutagenesis and DNA-mediated gene transfer. Using this technique, Ak beta polypeptides have been altered by substituting one or more Ad beta-specific residues at polymorphic positions in the beta 1 domain. Transfection of M12.C3 B lymphoma cells with most mutant Ak beta* genes results in the expression of Ak beta* Ad alpha molecules on the cell surface. However, the substitution of a single d allele residue at position 78 or 86 in the Ak beta polypeptide results in either the complete absence or very low levels, respectively, of cell surface expression of the Ak beta* Ad alpha molecule, but does not alter Ak beta* Ak alpha expression. The T.86 Ak beta* Ad alpha is expressed primarily in an intracellular compartment while the T.78 Ak beta* molecule does not appear to be produced. The core-glycosylated T.78 Ak beta* polypeptide does, however, form a complex intracellularly with the core-glycosylated Ii polypeptide. Substitution of the combination of d allele residues at Ak beta polymorphic positions 9, 12, 13, 14, and 17 results in the absence of Ak beta* Ak alpha cell surface expression but does not alter the expression of this mutant Ak beta* polypeptide with the Ad alpha polypeptide. These allele-specific expression mutants demonstrate that substitution at certain beta 1 domain positions may result in the alteration of Ia cell surface expression and that the transport of Ia molecules from the Golgi apparatus to the cell surface may be regulated by signals that are determined by the interaction of polymorphic residues in both the alpha and beta polypeptides.

ObjectiveTo identify neuromorphologic and neurochemical patterns of prenatal exposure to alcohol of developing brain of man as risk factor for development of mental pathology in offspring of mothers with alcoholism.MethodsWe studied brains of embryos and fetuses (7–12 weeks). There were obtained 53 embryos: 23 - from alcoholics and 30 - from healthy women. Age of patients was 26–39 years, disease duration - from 3 to 13 years. All cases were diagnosed with stage II alcoholism (F 10.201; F 10.202). To evaluate the properties of benzodiazepine receptors in brains of embryos sinaptoneurosomas we used method of radio receptor binding of selective ligand 3H-flyunitrazepame.ResultsDamage to cytoplasmic membrane and internal cell membrane systems, emergence and subsequent transformation of different types of spherical formations in perinuclear space, variety of options of mitochondria as a normal structure, and with signs of functional and structural pathology, increased development of Golgi complex, appearance of lipofuscin, multi-vesicular cells and myelin formation, delay of development of synaptic contacts of vesicular type, manifested in decreasing length of postsynaptic density of synaptic contacts, reducing area and perimeter of presynaptic terminals in main study group, changing nature of vascularization of brain tissue, what was expressed in reduction of average area of vessels, an increase in their number per unit of area and reducing perimeter of brain.ConclusionWe have found that alcoholism during pregnancy results in decrease of affinity of synaptosomal receptors of brains of embryos. This may affect overall neurotransmitter processes in brain.

ABSTRACTEhrlichia chaffeensisis an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions betweenE. chaffeensis47-kDa tandem repeat (TR) protein (TRP47) and the eukaryotic host cell have been described. In this investigation, yeast (Saccharomyces cerevisiae) two-hybrid analysis demonstrated thatE. chaffeensis-secreted tandem repeat protein 120 (TRP120) interacts with a diverse group of host cell proteins associated with major biological processes, including transcription and regulation, cell signaling, protein trafficking, and actin cytoskeleton organization. Twelve target proteins with the highest frequency of interaction with TRP120 were confirmed by cotransformation in yeast. Host targets, including human immunoglobulin lambda locus (IGL), cytochromecoxidase subunit II (COX2), Golgi-associated gamma adaptin ear-containing ARF binding protein 1 (GGA1), polycomb group ring finger 5 (PCGF5), actin gamma 1 (ACTG1), and unc-13 homolog D (UNC13D;Caenorhabditis elegans), colocalized strongly with TRP120 in HeLa cells and withE. chaffeensisdense-cored morulae and areas adjacent to morulae in the host cytoplasm. The TR domain of TRP120 interacted only with PCGF5, indicating that distinct TRP120 domains contribute to specific host target interactions and that multiple domains are required to reconstitute TRP120 interactions with other host targets. Three previously defined molecular interactions between TRP47 and host proteins, PCGF5, IGLL1, and CAP1, were also associated with TRP120, demonstrating that molecular cross talk occurs betweenEhrlichiaTRPs and host targets. These findings further support the role of TRPs as effectors that reprogram the host cell.