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European Journal of Biochemistry Jan 1995Vitamin-K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The...
Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity.
Vitamin-K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The APC-cofactor function of protein S is species specific, as human protein S potentiates the anticoagulant activity of human but not that of bovine APC, whereas bovine protein S is a cofactor to APC from both species. To elucidate which modules in protein S determine the species specificity, in vitro mutagenesis was used to construct six recombinant chimeric molecules between human and bovine protein S. Wild-type human and bovine protein S and the chimeras were expressed in 293 cells and the recombinant proteins purified by monoclonal antibody affinity chromatography. The recombinant proteins were found to be post-translationally modified, they bound C4b-binding protein and were functionally active as cofactors to APC. Chimeras having both the thrombin-sensitive region (TSR) and the first epidermal-growth-factor-(EGF)-like module of bovine origin expressed APC-cofactor activity similar to that of bovine protein S. Those chimeras, in which TSR or EGF1 derived from different species, manifested APC-cofactor activity similar to that of human protein S, i.e. they did not express cofactor activity to bovine APC. These data indicate that sequence differences in the TSR and EGF1 of human and bovine protein S cause the species specificity of the APC-cofactor activity. The data support the concept that these two modules of protein S interact with APC on the surface of negatively charged phospholipids.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Base Sequence; Cattle; Cell Line; Humans; Molecular Sequence Data; Oligodeoxyribonucleotides; Protein C; Protein S; Recombinant Fusion Proteins; Ribosomes; Sequence Homology, Amino Acid; Species Specificity
PubMed: 7851419
DOI: 10.1111/j.1432-1033.1995.tb20406.x -
The EMBO Journal Sep 1998Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomyces...
Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomyces cerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC). Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire. However, origins fire only upon the subsequent activation of S phase cyclin-dependent kinases (S-CDKs) and Dbf4/Cdc7 at the G1/S boundary. We have used a chromatin immunoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU). RPA's association with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm protein. The recruitment of DNA primase depends on RPA. Furthermore, early- and late-firing origins differ not in the timing of their recruitment of an Mcm protein, but in the timing of RPA's recruitment. RPA is recruited to early but not to late origins in HU. We also show that Rad53 kinase is required to prevent RPA association with a late origin in HU. Our data suggest that the origin unwinding accompanied by RPA association is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p. Thus, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open origins and thereby facilitate the loading of RPA.
Topics: Adenosine Triphosphatases; Cell Cycle Proteins; Checkpoint Kinase 2; Chromosomes, Fungal; Cyclin-Dependent Kinases; DNA Helicases; DNA Replication; DNA-Binding Proteins; Fungal Proteins; Hydroxyurea; Minichromosome Maintenance Complex Component 7; Nuclear Proteins; Protein Binding; Protein Kinases; Protein Serine-Threonine Kinases; Replication Origin; Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins
PubMed: 9724654
DOI: 10.1093/emboj/17.17.5182 -
Biochimica Et Biophysica Acta Dec 1995The congenital abnormal protein S(Tokushima) has Glu substituted for Lys155 in the second epidermal growth factor domain of the protein S molecule (Hayashi T., Nishioka...
The congenital abnormal protein S(Tokushima) has Glu substituted for Lys155 in the second epidermal growth factor domain of the protein S molecule (Hayashi T., Nishioka J., Shigekiyo, T. Saito, S. and Suzuki, K. (1994) Blood 83, 683-690). To elucidate the molecular mechanism of the dysfunction of the protein S(Tokushima), a comparative evaluation between the molecular interaction of the abnormal protein S and that of normal protein S with other clotting factors was carried out using recombinant normal protein S (rPSN) and protein S(Tokushima) (rPST) expressed in baby hamster kidney cells. While rPSN and plasma protein S exhibited cofactor activity for activated protein C (APC), rPST did not show this property. rPSN and rPST bound equally to phospholipids and C4b-binding protein fixed on microplate wells. APC bound to rPSN but not to rPST in an assay using immobilized monoclonal anti-protein S antibody. On the other hand, rPSN and plasma protein S inhibited the activity of prothrombinase complex composed of factor Xa and thrombin-stimulated platelets, whereas rPST lacked this inhibitory effect. Assessment of the mechanism by which rPST lacks inhibitory activity on the platelet-prothrombinase complex was also performed. Factor Xa bound to rPSN but not to rPST. Binding to rPSN to biotinylated factor Va in solution phase did not differ significantly from that of rPST. Binding of prothrombin to factor Va in solution phase was not inhibited either by rPSN or rPST. Binding of 4-amidinophenylmethanesulfonyl-factor Xa to factor Va in solution phase increased in the presence of rPSN but not in that of rPST. These findings suggest that the dysfunction of protein S(Tokushima) occurs because it fails to interact with APC and factor Xa. This molecular interaction is required for the expression of the APC cofactor activity and for the inhibition of the prothrombinase complex activity.
Topics: Amino Acid Sequence; Animals; Blood Coagulation; Cell Line; Cricetinae; Factor V; Factor Va; Factor X; Factor Xa; Humans; Molecular Sequence Data; Partial Thromboplastin Time; Phospholipids; Point Mutation; Protein C; Protein S; Prothrombin; Recombinant Proteins; Spectrometry, Fluorescence
PubMed: 8541347
DOI: 10.1016/0925-4439(95)00081-x -
FEBS Letters Aug 1995Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small... (Review)
Review
Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small GTPase Sar1p. The proteins that drive this cell-free vesicle budding reaction form an approximately 10 nm thick electron dense coat on ER-derived vesicles. Although the overall mechanism of membrane budding driven by various cytoplasmic coats appears similar, the constituents of this new membrane coat are molecularly distinct from the non-clathrin coat (COP) involved in intra-Golgi transport and the clathrin-containing coats. The new vesicle coat has been termed COPII.
Topics: Biological Transport; COP-Coated Vesicles; Endoplasmic Reticulum; Fungal Proteins; GTP-Binding Proteins; GTPase-Activating Proteins; Intracellular Membranes; Membrane Proteins; Monomeric GTP-Binding Proteins; Nuclear Pore Complex Proteins; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 7641893
DOI: 10.1016/0014-5793(95)00618-j -
Current Biology : CB Sep 2001The actin cytoskeleton has long been believed to play a role in endocytosis, but its actual function in this process has been unclear. Now, three proteins that promote... (Review)
Review
The actin cytoskeleton has long been believed to play a role in endocytosis, but its actual function in this process has been unclear. Now, three proteins that promote actin nucleation have been found to provide a link between the actin cytoskeleton and the endocytic machinery.
Topics: Actin-Related Protein 2; Actin-Related Protein 3; Actins; Animals; Cortactin; Cytoskeletal Proteins; Cytoskeleton; Endocytosis; Fungal Proteins; Microfilament Proteins; Proteins; Saccharomyces cerevisiae Proteins; Wiskott-Aldrich Syndrome Protein; Wiskott-Aldrich Syndrome Protein Family
PubMed: 11553341
DOI: 10.1016/s0960-9822(01)00410-9 -
Developmental Dynamics : An Official... Aug 2015Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous...
BACKGROUND
Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC, because the current knowledge regarding their expression is fragmentary or contradictory.
RESULTS
We first provide a comprehensive and detailed description of skeletal development in the cichlid fish Oreochromis mossambicus. Following this, we analyzed the expression pattern of OP-L and SPARC in detail during development. OP-L expression was only found in tissues that undergo ossification (i.e., developing bones and teeth). Furthermore, we show that there is a fundamental difference in cartilage formation of the splanchnocranium and all other cartilages, concerning SPARC expression. Significantly, we show that the initial calcification of cranial bones occurs simultaneously with the expression of OP-L and SPARC in the osteoblast-like cells, which appear early in development.
CONCLUSIONS
The difference in SPARC expression during chondrogenesis of the splanchnocranium is likely based on its different evolutionary history compared with the dermatocranium and chondrocranium. Moreover, our results suggest a co-occurrence of the initial calcium deposition and bone matrix protein expression during osteogenesis. Overall, this study enhances our understanding of fish skeletal development and evolution.
Topics: Animals; Cichlids; Fish Proteins; Osteonectin; Skeleton
PubMed: 25997670
DOI: 10.1002/dvdy.24293 -
Cell Dec 1997
Review
Topics: Animals; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Chromatin; DNA Replication; DNA-Binding Proteins; Models, Genetic; Nuclear Proteins; Replication Origin; Saccharomyces cerevisiae Proteins; Xenopus
PubMed: 9413980
DOI: 10.1016/s0092-8674(00)80459-6 -
The Journal of Biological Chemistry Apr 2011Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork...
Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork helicase in eukaryotes. We purified Sld3, Cdc45, GINS, and Mcm2-7 and studied their interaction and assembly into complexes. Sld3 binds tightly to Cdc45 in the presence or absence of cyclin-dependent kinase activity. Furthermore, Sld3 binds tightly to the Mcm2-7 complex, and a ternary complex forms among Cdc45, Mcm2-7, and Sld3, with a 1:1:1 stoichiometry (CMS complex). GINS binds directly to Mcm2-7, and GINS competes with Sld3 for Mcm2-7 binding. GINS also binds directly to Cdc45, and GINS competes with Sld3 for Cdc45 binding. Cdc45, Mcm2-7, and GINS form a ternary complex with a stoichiometry of 1:1:1 (CMG complex). Size exclusion data reveal that when Sld3, Cdc45, Mcm2-7, and GINS are added together, the result is a mixture of CMS and CMG complexes. The data suggest that GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding. Our results are consistent with a model wherein GINS trades places with Sld3 at a replication origin, contributing to the activation of the replication fork helicase.
Topics: Binding, Competitive; Cell Cycle; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; DNA Replication; DNA-Binding Proteins; Gene Expression Regulation, Fungal; Glutathione Transferase; Nuclear Proteins; Protein Binding; Replication Origin; Ribonucleoprotein, U4-U6 Small Nuclear; Ribonucleoprotein, U5 Small Nuclear; Saccharomyces cerevisiae Proteins; Saccharomycetales
PubMed: 21362622
DOI: 10.1074/jbc.M111.218305 -
The Journal of Cell Biology Sep 1996
Review
Topics: Cell Cycle Proteins; Cytoskeletal Proteins; Cytoskeleton; Fungal Proteins; GTP Phosphohydrolases; Membrane Proteins; Saccharomyces cerevisiae Proteins; Schizosaccharomyces pombe Proteins; Transcription Factors
PubMed: 8830765
DOI: 10.1083/jcb.134.6.1345 -
Redox Biology Oct 2020Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species...
Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress.
Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite. For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs. Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.
Topics: Air Pollution; Humans; NF-kappa B; Oxidative Stress; Peroxynitrous Acid; Toll-Like Receptor 4
PubMed: 32739154
DOI: 10.1016/j.redox.2020.101581