RESUMEN
ING2 (inhibitor of growth 2) is a candidate tumor-suppressor gene involved in cell cycle control, apoptosis and senescence. Although the functions of ING2 within the chromatin remodeling complex Sin3A/histone deacetylase (HDAC) and in the p53 pathway have been described, how ING2 itself is regulated remains unknown. In this study we report for the first time that ING2 can be sumoylated by small ubiquitin-like modifier 1 (SUMO1) on lysine 195 both in vitro and in vivo. Strikingly, ING2 sumoylation enhances its association with Sin3a. We provide evidences that ING2 can bind to the promoter of genes to mediate their expression and that sumoylation of ING2 is required for this binding to some of these genes. Among them, we identified the gene TMEM71 (transmembrane protein 71), whose expression is regulated by ING2 sumoylation. ING2 must be sumoylated to bind to the promoter of TMEM71 and to recruit the Sin3A chromatin-modifying complex to this promoter, in order to regulate TMEM71 transcription. Hence, sumoylation of ING2 enhances its binding to the Sin3A/HDAC complex and is required to regulate gene transcriptions.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Represoras/fisiología , Transcripción Genética/fisiología , Proteínas Supresoras de Tumor/fisiología , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Proteínas de Homeodominio/metabolismo , Humanos , Unión Proteica , ARN Interferente Pequeño , Receptores Citoplasmáticos y Nucleares/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3 , Fracciones Subcelulares/metabolismo , Sumoilación , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Système pour l'Observation de la Terre images are used to map ground displacements induced by earthquakes. Deformations (offsets) induced by stereoscopic effect and roll, pitch, and yaw of satellite and detector artifacts are estimated and compensated. Images are then resampled in a cartographic projection with a low-bias interpolator. A subpixel correlator in the Fourier domain provides two-dimensional offset maps with independent measurements approximately every 160 m. Biases on offsets are compensated from calibration. High-frequency noise (0.125 m(-1)) is approximately 0.01 pixels. Low-frequency noise (lower than 0.001 m(-1)) exceeds 0.2 pixels and is partially compensated from modeling. Applied to the Landers earthquake, measurements show the fault with an accuracy of a few tens of meters and yields displacement on the fault with an accuracy of better than 20 cm. Comparison with a model derived from geodetic data shows that offsets bring new insights into the faulting process.
RESUMEN
Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Hemoproteínas/metabolismo , Lipasa/metabolismo , Proteínas de Transporte de Membrana , Metaloendopeptidasas/metabolismo , Serratia marcescens/enzimología , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Colagenasas/genética , Colagenasas/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Proteínas de Unión al Hemo , Hemoproteínas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Serratia marcescens/genéticaRESUMEN
One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells is sec-independent and dedicated to proteins lacking an N-terminal signal peptide. Most of these proteins display a C-terminal secretion signal located in the last 60 amino acids (aa). Using one Erwinia chrysanthemi protease, PrtG, secreted by such a pathway it was shown that the smallest C-terminal sequence allowing efficient secretion contains the last 29 aa of PrtG and that low but significant secretion can be promoted by the last 15 aa of PrtG. Moreover, the extreme C-terminal motif, consisting of a negatively charged aa followed by several hydrophobic aa must be exposed and is conserved amongst many proteins following this pathway. This secretion system depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. These Gram-negative bacterial protein exporters are dedicated to the secretion of one or several closely related proteins belonging to the toxin, protease and lipase families. The genes encoding the three secretion proteins and the exoproteins are usually all linked, consistent with the specificity of the systems. Er. chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on protease and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three components' association was ordered and substrate binding was required for the formation of this multiprotein complex.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras , Bacterias Gramnegativas/metabolismo , Transporte Biológico , Dickeya chrysanthemi/química , Dickeya chrysanthemi/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Señales de Clasificación de Proteína/química , Proteínas Recombinantes de Fusión/metabolismo , Serratia marcescens/química , Serratia marcescens/metabolismoRESUMEN
One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins: an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteinases B and C, and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on proteinase and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter/secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three component association was ordered and substrate binding was required for the formation of this multiprotein complex.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico , Membrana Celular/metabolismo , Señales de Clasificación de Proteína/metabolismoRESUMEN
The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli, TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi. By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens. hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras , Genes Bacterianos , Serratia marcescens/genética , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Prueba de Complementación Genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Serratia marcescens/metabolismoRESUMEN
The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous although the secreted polypeptides share no sequence homology. Whereas protease C can use both translocators, HasA is secreted only by its specific transporter. Functional analysis of protease C and HasA secretion through hybrid transporters obtained by combining components from each system demonstrates that the ABC-protein is responsible for the substrate specificity and that inhibition of protease C secretion in the presence of HasA results from a defective interaction between HasA and the ABC-protein. We also show that the outer membrane protein, TolC, can combine with the membrane fusion protein HasE in the presence of either ABC-protein to form a functional transporter but not with the membrane fusion protein, PrtE. This indicates a specific interaction between the outer membrane component and the membrane fusion protein.