ABSTRACT
The centromere is essential for ensuring high-fidelity transmission of chromosomes. CENP-A, the centromeric histone H3 variant, is thought to be the epigenetic mark of centromere identity. CENP-A deposition at the centromere is crucial for proper centromere function and inheritance. Despite its importance, the precise mechanism responsible for maintenance of centromere position remains obscure. Here, we report a mechanism to maintain centromere identity. We demonstrate that CENP-A interacts with EWSR1 (Ewing sarcoma breakpoint region 1) and EWSR1-FLI1 (the oncogenic fusion protein in Ewing sarcoma). EWSR1 is required for maintaining CENP-A at the centromere in interphase cells. EWSR1 and EWSR1-FLI1 bind CENP-A through the SYGQ2 region within the prion-like domain, important for phase separation. EWSR1 binds to R-loops through its RNA-recognition motif in vitro. Both the domain and motif are required for maintaining CENP-A at the centromere. Therefore, we conclude that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA.
Subject(s)
Centromere , RNA-Binding Protein EWS , Humans , Autoantigens/metabolism , Centromere/metabolism , Centromere Protein A/genetics , Chromosomal Proteins, Non-Histone/metabolism , RNA , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , Sarcoma, EwingABSTRACT
Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem ß-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal ß-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Cytotoxins/chemistry , Mycoplasma pneumoniae/metabolism , Vacuoles/metabolism , 1,2-Dipalmitoylphosphatidylcholine/metabolism , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/metabolism , Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Catalytic Domain , Cytotoxins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Phosphatidylcholines/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Sphingomyelins/metabolism , Structure-Activity RelationshipABSTRACT
Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591-amino-acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modelling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.
Subject(s)
ADP Ribose Transferases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Mycoplasma pneumoniae , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/genetics , Amino Acid Motifs , Animals , Bacterial Proteins/genetics , Bacterial Toxins/genetics , HeLa Cells , Humans , Models, Molecular , NAD/metabolism , Protein Structure, Tertiary , Proteolysis , Virulence Factors/chemistry , Virulence Factors/metabolismABSTRACT
Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 A, beta = 90.6 degrees. A complete 2.2 A data set was obtained from a single CARDS TX crystal.
