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Journal of Thrombosis and Haemostasis :... Dec 2019Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and...
BACKGROUND
Activated protein C (APC)-mediated inactivation of factor (F)Va is greatly enhanced by protein S. For inactivation to occur, a trimolecular complex among FVa, APC, and protein S must form on the phospholipid membrane. However, direct demonstration of complex formation has proven elusive.
OBJECTIVES
To elucidate the nature of the phospholipid-dependent interactions among APC, protein S, and FVa.
METHODS
We evaluated binding of active site blocked APC to phospholipid-coated magnetic beads in the presence and absence of protein S and/or FVa. The importance of protein S and FV residues were evaluated functionally.
RESULTS
Activated protein C alone bound weakly to phospholipids. Protein S mildly enhanced APC binding to phospholipid surfaces, whereas FVa did not. However, FVa together with protein S enhanced APC binding (>14-fold), demonstrating formation of an APC/protein S/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding, agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A) with reduced APC cofactor function exhibited essentially normal augmentation of APC binding to phospholipids, but diminished APC/protein S/FVa complex formation, suggesting involvement in interactions dependent upon FVa. Similarly, FVa (W1920R), an APC-resistant FV variant, also did not efficiently incorporate into the trimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggested that the mutation impairs its affinity for phospholipid membranes and with protein S within the complex.
CONCLUSIONS
FVa plays a central role in the formation of its inactivation complex. Furthermore, membrane proximal interactions among FVa, APC, and protein S are essential for its cofactor function.
Topics: Binding Sites; Blood Coagulation; Calcium-Binding Proteins; Enzyme Activation; Factor Va; HEK293 Cells; Humans; Models, Molecular; Multiprotein Complexes; Phospholipids; Protein Binding; Protein C; Protein Conformation; Protein S; Structure-Activity Relationship; Thrombin; Thromboplastin
PubMed: 31364267
DOI: 10.1111/jth.14594 -
European Journal of Biochemistry Feb 1990Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was...
Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 micrograms/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2(+)-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide-gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.
Topics: Amino Acid Sequence; Amino Acids; Animals; DNA; Electrophoresis, Polyacrylamide Gel; Gene Expression; Genetic Vectors; Glycoproteins; Glycoside Hydrolases; Humans; Mice; Molecular Sequence Data; Plasmids; Protein Processing, Post-Translational; Protein S; Recombinant Proteins; Structure-Activity Relationship; Transfection
PubMed: 2137411
DOI: 10.1111/j.1432-1033.1990.tb15361.x -
Cell Cycle (Georgetown, Tex.) Oct 2013
Topics: Autophagy-Related Proteins; GTP Phosphohydrolases; Gene Deletion; Mitochondria; Mitochondrial Proteins; Mitophagy; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 24013417
DOI: 10.4161/cc.26352 -
Philosophical Transactions of the Royal... Aug 1999The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins... (Review)
Review
The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins move on their way to or from the cell surface. Sorting of proteins within this system is achieved by their selective incorporation into budding vesicles and the specific fusion of these with an appropriate target membrane. Cytosolic coat proteins help to select vesicle contents, while fusion is mediated by membrane proteins termed SNAREs present in both vesicles and target membranes. SNAREs are not the sole determinants of target specificity, but they lie at the heart of the fusion process. The complete set of SNAREs is known in yeast, and analysis of their locations, interactions and functions in vivo gives a comprehensive picture of the traffic routes and the ways in which organelles such as the Golgi apparatus are formed. The principles of protein and lipid sorting revealed by this analysis are likely to apply to a wide variety of eukaryotic cells.
Topics: Adenosine Triphosphatases; Biological Transport; Biomarkers; Endocytosis; Fungal Proteins; Golgi Apparatus; Intracellular Membranes; Membrane Fusion; Membrane Proteins; Membrane Transport Proteins; Nerve Tissue Proteins; Qa-SNARE Proteins; SNARE Proteins; Saccharomyces cerevisiae Proteins; Synaptosomal-Associated Protein 25; Vesicular Transport Proteins
PubMed: 10515003
DOI: 10.1098/rstb.1999.0491 -
The Journal of Biological Chemistry Sep 1986Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding...
Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.
Topics: Animals; Carrier Proteins; Cattle; Complement Inactivator Proteins; Factor V; Factor Va; Glycoproteins; Humans; Immunosorbent Techniques; Protein C; Protein S
PubMed: 2943733
DOI: No ID Found -
Current Biology : CB Sep 1999In many cells, centrosomes are required to position nuclei at specific locations in the cytoplasm. The nature of the link between centrosomes and nuclei is mysterious,... (Review)
Review
In many cells, centrosomes are required to position nuclei at specific locations in the cytoplasm. The nature of the link between centrosomes and nuclei is mysterious, but the recently characterised UNC84 protein appears to be involved.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Cycle Proteins; Cell Nucleus; Centrosome; Dyneins; Eukaryotic Cells; Fungal Proteins; Helminth Proteins; Membrane Glycoproteins; Molecular Motor Proteins; Nuclear Proteins; Proteasome Endopeptidase Complex; RNA-Binding Proteins; Recombinant Fusion Proteins; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 10508607
DOI: 10.1016/s0960-9822(99)80446-1 -
Seminars in Cell & Developmental Biology Jul 2011Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the... (Review)
Review
Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we summarize the characteristics of each prion element, and discuss their potential functional roles in yeast biology.
Topics: Amyloid; Caspases; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Epigenomics; Glutathione Peroxidase; Peptide Termination Factors; Prions; Proton-Translocating ATPases; Repressor Proteins; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcription Factors
PubMed: 21397710
DOI: 10.1016/j.semcdb.2011.03.003 -
Journal of Virology May 2022As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young...
As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry. As the most important nonpolio neurotropic enterovirus lacking specific treatments, EV71 can transmit to the central nervous system, leading to severe and fatal neurological complications in infants and young children. The identification of new factors that facilitate or inhibit EV71 replication is crucial to uncover the mechanisms of viral infection and pathogenesis. To date, only a few host factors involved in EV71 infection have been characterized. Herein, we conducted a genome-wide CRISPR-Cas9 functional knockout (GeCKO) screen for the first time to study EV71 in HeLa cells. The screening results are presented as a ranked list of candidates, including 518 hits in the positive selection that facilitate EV71 replication and 1,044 hits in the negative selection that may be essential for cell growth and survival or for suppressing EV71 infection. We subsequently concentrated on the top three hits in the positive selection: SCARB2, SLC35B2, and B3GAT3. The knockout of any of these three genes confers strong resistance against EV71 infection. We confirmed that EV71 infection is codependent on two receptors, heparan sulfate and SCARB2. We also identified a host entry factor, SLC35B2, indirectly facilitating EV71 infection through regulation of the host cell sulfation, and determined a novel posttranslational modification, protein tyrosine sulfation existing in SCARB2. This study revealed that EV71 infectivity exhibits a significant positive correlation with the level of cellular sulfation regulated by SLC35B2. Due to the sulfation pathway being required for many distinct viruses, including but not limited to EV71 and respiratory syncytial virus (RSV), which were tested in this study, SLC35B2 represents a target of broad-spectrum antiviral therapy.
Topics: Enterovirus A, Human; Enterovirus Infections; Glucuronosyltransferase; HeLa Cells; Heparitin Sulfate; Humans; Lysosomal Membrane Proteins; Receptors, Scavenger; Sulfate Transporters; Sulfotransferases; Tyrosine
PubMed: 35420441
DOI: 10.1128/jvi.02042-21 -
Molecular Biology of the Cell Dec 1999Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and...
Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.
Topics: Exocytosis; Fungal Proteins; Golgi Apparatus; Membrane Proteins; Mutation; Protein Binding; Qa-SNARE Proteins; Qc-SNARE Proteins; R-SNARE Proteins; SNARE Proteins; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins; Yeasts; rab GTP-Binding Proteins
PubMed: 10588649
DOI: 10.1091/mbc.10.12.4149 -
The Journal of Biological Chemistry Oct 1989Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a...
Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.
Topics: Amino Acid Sequence; Carrier Proteins; Complement C4; Complement Inactivator Proteins; Glycoproteins; Humans; Kinetics; Molecular Sequence Data; Oligopeptides; Protein S; Receptors, Complement; Sequence Homology, Nucleic Acid
PubMed: 2530213
DOI: No ID Found