RESUMEN
Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.
Asunto(s)
Neurofibromatosis 1 , Neurofibromina 1 , Microscopía por Crioelectrón , Proteínas Activadoras de GTPasa/metabolismo , Genes de Neurofibromatosis 1 , Humanos , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Neurofibromina 1/química , Neurofibromina 1/genética , Neurofibromina 1/metabolismoRESUMEN
Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores ErbB/metabolismo , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/química , Secuencia de Aminoácidos , Manchas Café con Leche/genética , Dominio Catalítico , Análisis Mutacional de ADN , Factor de Crecimiento Epidérmico/farmacología , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Células K562 , Neurofibromatosis 1/genética , Neurofibromina 1/genética , Fosforilación , Mutación Puntual/genética , Unión Proteica , Dominios Proteicos , Mapas de Interacción de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de SeñalRESUMEN
About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.
Asunto(s)
Glicósido Hidrolasas/genética , Hojas de la Planta/enzimología , Zea mays/enzimología , beta-Fructofuranosidasa/genética , Secuencia de Aminoácidos , Metabolismo de los Hidratos de Carbono/genética , Fructanos/genética , Fructanos/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Glicósido Hidrolasas/química , Hojas de la Planta/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Especificidad por Sustrato , Nicotiana/enzimología , Nicotiana/genética , Zea mays/genética , beta-Fructofuranosidasa/antagonistas & inhibidoresRESUMEN
Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10-6â M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (KM, Vmax) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glutamato-Cisteína Ligasa/química , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/biosíntesis , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Dominio Catalítico , Activación Enzimática , Glutamato-Cisteína Ligasa/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Plantas Modificadas Genéticamente , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Ras-specific GTPase-activating proteins (RasGAPs) down-regulate the biological activity of Ras proteins by accelerating their intrinsic rate of GTP hydrolysis, basically by a transition state stabilizing mechanism. Oncogenic Ras is commonly not sensitive to RasGAPs caused by interference of mutants with the electronic or steric requirements of the transition state, resulting in up-regulation of activated Ras in respective cells. RasGAPs are modular proteins containing a helical catalytic RasGAP module surrounded by smaller domains that are frequently involved in the subcellular localization or contributing to regulatory features of their host proteins. In this review, we summarize current knowledge about RasGAP structure, mechanism, regulation, and dual-substrate specificity and discuss in some detail neurofibromin, one of the most important negative Ras regulators in cellular growth control and neuronal function.
Asunto(s)
Proteínas Activadoras de ras GTPasa/química , Aumento de la Célula , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , GTP Fosfohidrolasas/metabolismo , Uniones Comunicantes/fisiología , Humanos , Estructura Molecular , Neurofibromina 1/fisiología , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/fisiologíaRESUMEN
Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible 'lid'-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes.
Asunto(s)
Aminoácidos/metabolismo , Carboxiliasas/metabolismo , Hidrolasas/metabolismo , Proteínas Mitocondriales/metabolismo , Aminoácidos/química , Aminoácidos/genética , Carboxiliasas/química , Carboxiliasas/genética , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Hidrolasas/química , Hidrolasas/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Modelos Moleculares , Mutación , Conformación Proteica , Piruvatos/química , Piruvatos/metabolismo , Especificidad por SustratoRESUMEN
MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.
Asunto(s)
Cromatina/genética , Epigénesis Genética/genética , Histonas/química , Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/genética , Cromatina/química , Cristalografía por Rayos X , Daño del ADN/genética , Heterocromatina/química , Heterocromatina/genética , Histonas/genética , Humanos , Poli(ADP-Ribosa) Polimerasa-1/química , Poli(ADP-Ribosa) Polimerasa-1/genética , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genéticaRESUMEN
Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.
Asunto(s)
Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Proteínas Portadoras/química , Glicoproteínas/química , Fragmentos Fab de Inmunoglobulinas/química , Procesamiento Proteico-Postraduccional , Albúmina Sérica Humana/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismoRESUMEN
Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Portadoras/genética , Endosomas/efectos de los fármacos , Endosomas/ultraestructura , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Movimiento , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Transducción de SeñalRESUMEN
The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases.
Asunto(s)
Proteínas Portadoras/química , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Portadoras/ultraestructura , Cristalografía por Rayos X , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/ultraestructura , Humanos , Péptidos y Proteínas de Señalización Intracelular , Dominios Proteicos , Transducción de SeñalRESUMEN
Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive ß-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Neurofibromina 1/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Adaptadoras Transductoras de Señales , Dominios ProteicosRESUMEN
The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the VL1 antigen-binding loop, with the α-1-6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. In addition, the map quality at 1.9â Å resolution was sufficient to crystallographically re-sequence the variable VL and VH domains and to detect discrepancies in the hybridoma-derived sequence. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Proteínas Portadoras/inmunología , Glicoproteínas/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Albúmina Sérica/inmunología , Animales , Complejo Antígeno-Anticuerpo , Regiones Determinantes de Complementariedad , Cristalografía por Rayos X , Glicosilación , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Ratones , Modelos Moleculares , Albúmina Sérica HumanaRESUMEN
Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neurofibromina 1/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Células HEK293 , Humanos , Dominios ProteicosRESUMEN
The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5â mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1â M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1â M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity.
Asunto(s)
Péptido Hidrolasas/química , Inhibidores de Proteasas/química , Automatización de Laboratorios , Cristalización , Cristalografía por Rayos X , Contaminación de Equipos , RobóticaAsunto(s)
Genes MHC Clase II/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Neurofibromina 1/deficiencia , Células de Schwann/metabolismo , Regulación hacia Arriba/fisiología , Línea Celular Tumoral , Células Cultivadas , Genes MHC Clase II/genética , Antígenos HLA-DP/metabolismo , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Neurofibroma , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Nervios Periféricos/metabolismo , Nervios Periféricos/patología , Fenotipo , Células de Schwann/patología , Regulación hacia Arriba/genéticaRESUMEN
De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.
Asunto(s)
Trastorno Autístico/genética , Epilepsia/genética , Haploinsuficiencia , Discapacidad Intelectual/genética , Proteínas Activadoras de ras GTPasa/genética , Adolescente , Secuencia de Aminoácidos , Trastorno Autístico/fisiopatología , Western Blotting , Niño , Preescolar , Clonación Molecular , Epilepsia/fisiopatología , Exoma , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Células HEK293 , Humanos , Discapacidad Intelectual/fisiopatología , Masculino , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Fosforilación , Conformación Proteica , Análisis de Secuencia de ADN , Transfección , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
The initial reports on pleckstrin homology (PH) domains almost 20 years ago described them as sequence feature of proteins involved in signal transduction processes. Investigated at first along the phospholipid binding properties of a small subset of PH representatives, the PH fold turned out to appear as mediator of phosphotyrosine and polyproline peptide binding to other signaling proteins. While phospholipid binding now seems rather the exception among PH-like domains, protein-protein interactions established as more and more important feature of these modules. In this review we focus on the PH superfold as a versatile protein-protein interaction platform and its three-dimensional integration in an increasing number of available multidomain structures.
Asunto(s)
Proteínas Sanguíneas/química , Fosfoproteínas/química , Mapeo de Interacción de Proteínas/métodos , Estructura Terciaria de Proteína , Animales , Cristalografía por Rayos X/métodos , Proteínas de Unión al GTP/química , Guanosina Trifosfato/química , Humanos , Ligandos , Modelos Moleculares , Péptidos/química , Fosfolípidos/química , Conformación Proteica , Pliegue de Proteína , Transducción de SeñalRESUMEN
Physiological processes are strictly organized in space and time. However, in cell physiology research, more attention is given to the question of space rather than to time. To function as a signal, environmental changes must be restricted in time; they need not only be initiated but also terminated. In this review, we concentrate on the role of one specific protein family involved in biological signal termination. GTPase activating proteins (GAPs) accelerate the endogenously low GTP hydrolysis rate of monomeric guanine nucleotide-binding proteins (GNBPs), limiting thereby their prevalence in the active, GTP-bound form. We discuss cases where defective or excessive GAP activity of specific proteins causes significant alteration in the function of the nervous, endocrine, and hemopoietic systems, or contributes to development of infections and tumors. Biochemical and genetic data as well as observations from human pathology support the notion that GAPs represent vital elements in the spatiotemporal fine tuning of physiological processes.
Asunto(s)
Sistema Endocrino/fisiología , Proteínas Activadoras de GTPasa/fisiología , Hematopoyesis/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Transformación Celular Neoplásica , Humanos , Infecciones/fisiopatologíaRESUMEN
Plants and bacteria assimilate sulfur into cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase complex (CSC), which consists of serine-acetyl-transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL) enzymes. The activity of OAS-TL is reduced by formation of the CSC. Although this reduction is an inherent part of the self-regulation cycle of cysteine biosynthesis, there has until now been no explanation as to how OAS-TL loses activity in plants. Complexation of SAT and OAS-TL involves binding of the C-terminal tail of SAT in one of the active sites of the homodimeric OAS-TL. We here explore the flexibility of the unoccupied active site in Arabidopsis thaliana cytosolic and mitochondrial OAS-TLs. Our results reveal two gates in the OAS-TL active site that define its accessibility. The observed dynamics of the gates show allosteric closure of the unoccupied active site of OAS-TL in the CSC, which can hinder substrate binding, abolishing its turnover to cysteine.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Cisteína Sintasa/química , Cisteína/biosíntesis , Mitocondrias/enzimología , Regulación Alostérica , Secuencias de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Unión Proteica , Estructura Cuaternaria de Proteína , Serina O-Acetiltransferasa/químicaRESUMEN
Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.