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1.
Methods Mol Biol ; 2705: 77-89, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37668970

RESUMEN

The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.


Asunto(s)
Fosfopéptidos , Proteína Activadora de GTPasa p120 , Cristalografía por Rayos X , Fosfotirosina , Difracción de Rayos X
2.
J Biol Chem ; 299(9): 105098, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37507023

RESUMEN

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.


Asunto(s)
Proteínas Tirosina Quinasas Receptoras , Proteína Activadora de GTPasa p120 , Humanos , Proteínas Activadoras de GTPasa/metabolismo , Proteína Activadora de GTPasa p120/química , Fosforilación , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Dominios Homologos src , Calorimetría , Péptidos/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño
3.
Am J Hum Genet ; 110(5): 846-862, 2023 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-37086723

RESUMEN

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p = 4.83 × 10-20) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance > 0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p = 1.1 × 10-11). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling.


Asunto(s)
Craneosinostosis , Tretinoina , Humanos , Mutación , Craneosinostosis/genética , Regulación de la Expresión Génica , Cromatina , Predisposición Genética a la Enfermedad
4.
Hum Genet ; 142(1): 21-32, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-35997807

RESUMEN

Lambdoid craniosynostosis (CS) is a congenital anomaly resulting from premature fusion of the cranial suture between the parietal and occipital bones. Predominantly sporadic, it is the rarest form of CS and its genetic etiology is largely unexplored. Exome sequencing of 25 kindreds, including 18 parent-offspring trios with sporadic lambdoid CS, revealed a marked excess of damaging (predominantly missense) de novo mutations that account for ~ 40% of sporadic cases. These mutations clustered in the BMP signaling cascade (P = 1.6 × 10-7), including mutations in genes encoding BMP receptors (ACVRL1 and ACVR2A), transcription factors (SOX11, FOXO1) and a transcriptional co-repressor (IFRD1), none of which have been implicated in other forms of CS. These missense mutations are at residues critical for substrate or target sequence recognition and many are inferred to cause genetic gain-of-function. Additionally, mutations in transcription factor NFIX were implicated in syndromic craniosynostosis affecting diverse sutures. Single cell RNA sequencing analysis of the mouse lambdoid suture identified enrichment of mutations in osteoblast precursors (P = 1.6 × 10-6), implicating perturbations in the balance between proliferation and differentiation of osteoprogenitor cells in lambdoid CS. The results contribute to the growing knowledge of the genetics of CS, have implications for genetic counseling, and further elucidate the molecular etiology of premature suture fusion.


Asunto(s)
Craneosinostosis , Ratones , Animales , Craneosinostosis/genética , Craneosinostosis/metabolismo , Mutación , Transducción de Señal/genética , Factores de Transcripción/genética , Diferenciación Celular , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo
5.
Structure ; 30(12): 1603-1614.e5, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36417908

RESUMEN

p120RasGAP is a multidomain GTPase-activating protein for Ras. The presence of two Src homology 2 domains in an SH2-SH3-SH2 module raises the possibility that p120RasGAP simultaneously binds dual phosphotyrosine residues in target proteins. One known binding partner with two proximal phosphotyrosines is p190RhoGAP, a GTPase-activating protein for Rho GTPases. Here, we present the crystal structure of the p120RasGAP SH2-SH3-SH2 module bound to a doubly tyrosine-phosphorylated p190RhoGAP peptide, revealing simultaneous phosphotyrosine recognition by the SH2 domains. The compact arrangement places the SH2 domains in close proximity resembling an SH2 domain tandem and exposed SH3 domain. Affinity measurements support synergistic binding, while solution scattering reveals that dual phosphotyrosine binding induces compaction of this region. Our studies reflect a binding mode that limits conformational flexibility within the SH2-SH3-SH2 cassette and relies on the spacing and sequence surrounding the two phosphotyrosines, potentially representing a selectivity mechanism for downstream signaling events.


Asunto(s)
Proteína Activadora de GTPasa p120 , Dominios Homologos src , Fosfotirosina , Proteínas Activadoras de GTPasa , Tirosina
6.
Nat Commun ; 13(1): 4788, 2022 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-35970859

RESUMEN

RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.


Asunto(s)
Proteína Activadora de GTPasa p120 , Dominios Homologos src , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Activadora de GTPasa p120/química , Proteína Activadora de GTPasa p120/genética , Proteína Activadora de GTPasa p120/metabolismo , Proteínas de Unión al GTP rho/metabolismo
7.
J Exp Med ; 218(7)2021 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-33951726

RESUMEN

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.


Asunto(s)
Agammaglobulinemia/genética , Cromatina/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Adolescente , Adulto , Linfocitos B/fisiología , Diferenciación Celular/genética , Línea Celular , Niño , Preescolar , Células Dendríticas/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Lactante , Linfopoyesis/genética , Masculino , Mutación/genética , Células Precursoras de Linfocitos B/fisiología , Células Madre/fisiología , Adulto Joven
8.
FEBS J ; 287(19): 4232-4245, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32893973

RESUMEN

Pseudoenzymes are emerging as significant mediators and regulators of signal transduction. These proteins maintain enzyme folds and topologies, but are disrupted in the conserved motifs required for enzymatic activity. Among the pseudoenzymes, the pseudoGTPase group of atypical GTPases has recently expanded and includes the Rnd and RGK groups, RhoH and the RhoBTB proteins, mitochondrial RhoGTPase and centaurin-γ groups, CENP-M, dynein LIC, Entamoeba histolytica RabX3, leucine-rich repeat kinase 2, and the p190RhoGAP proteins. The wide range of cellular functions associated with pseudoGTPases includes cell migration and adhesion, membrane trafficking and cargo transport, mitosis, mitochondrial activity, transcriptional control, and autophagy, placing the group in an expanding portfolio of signaling pathways. In this review, we examine how the pseudoGTPases differ from canonical GTPases and consider their mechanistic and functional roles in signal transduction. We review the amino acid differences between the pseudoGTPases and discuss how these proteins can be classified based on their ability to bind nucleotide and their enzymatic activity. We discuss the molecular and structural consequences of amino acid divergence from canonical GTPases and use comparison with the well-studied pseudokinases to illustrate the classifications. PseudoGTPases are fast becoming recognized as important mechanistic components in a range of cellular roles, and we provide a concise discussion of the currently identified members of this group. ENZYMES: small GTPases; EC number: EC 3.6.5.2.


Asunto(s)
GTP Fosfohidrolasas , Proteínas/metabolismo , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/genética , Transducción de Señal
9.
J Biol Chem ; 295(31): 10511-10521, 2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32540970

RESUMEN

The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved FLVR motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket that appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position ßD4) and K400A (SH2 position ßD6) is required to disrupt the binding. These results indicate a hitherto unrecognized diversity in SH2 domain interactions with phosphotyrosine and classify the C-terminal SH2 domain of p120RasGAP as "FLVR-unique."


Asunto(s)
Evolución Molecular , Proteína Activadora de GTPasa p120/química , Cristalografía por Rayos X , Humanos , Proteína Activadora de GTPasa p120/genética , Proteína Activadora de GTPasa p120/metabolismo , Dominios Homologos src
10.
PLoS One ; 15(2): e0229627, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32078652

RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0226113.].

11.
Proc Natl Acad Sci U S A ; 116(45): 22730-22736, 2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31624127

RESUMEN

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/genética , Secuenciación del Exoma , Mutación , Receptor ErbB-2/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/terapia , Animales , Línea Celular Tumoral , Terapia Combinada , Variaciones en el Número de Copia de ADN , Femenino , Xenoinjertos , Humanos , Polimorfismo de Nucleótido Simple , Neoplasias del Cuello Uterino/patología
12.
Transl Res ; 208: 15-29, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30802431

RESUMEN

Arrhythmogenic cardiomyopathy is a genetic heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes leading to life-threatening ventricular arrhythmias, heart failure, and sudden cardiac death. Mutations in genes encoding cardiac junctional proteins are known to cause about half of cases, while remaining genetic causes are unknown. Using exome sequencing, we identified 2 missense variants (p.H33N and p.H77Y) that were predicted to be damaging in the integrin-linked kinase (ILK) gene in 2 unrelated families. The p.H33N variant was found to be de novo. ILK links integrins and the actin cytoskeleton, and is essential for the maintenance of normal cardiac function. Both of the new variants are located in the ILK ankyrin repeat domain, which binds to the first LIM domain of the adaptor proteins PINCH1 and PINCH2. In silico binding studies proposed that the human variants disrupt the ILK-PINCH complex. Recombinant mutant ILK expressed in H9c2 rat myoblast cells shows aberrant prominent cytoplasmic localization compared to the wild-type. Expression of human wild-type and mutant ILK under the control of the cardiac-specific cmlc2 promotor in zebrafish shows that p.H77Y and p.P70L, a variant previously reported in a dilated cardiomyopathy family, cause cardiac dysfunction and death by about 2-3 weeks of age. Our findings provide genetic and functional evidence that ILK is a cardiomyopathy disease gene and highlight its relevance for diagnosis and genetic counseling of inherited cardiomyopathies.


Asunto(s)
Arritmias Cardíacas/genética , Cardiomiopatías/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Adolescente , Secuencia de Aminoácidos , Animales , Línea Celular , Femenino , Humanos , Masculino , Mutación Missense , Linaje , Proteínas Serina-Treonina Quinasas/química , Ratas , Homología de Secuencia de Aminoácido , Secuenciación del Exoma , Pez Cebra/genética
13.
PLoS One ; 14(12): e0226113, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31891593

RESUMEN

The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100-1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the ßE-ßF loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 µM between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.


Asunto(s)
Fosfopéptidos/química , Proteína Activadora de GTPasa p120/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Mutagénesis Sitio-Dirigida , Fosfopéptidos/síntesis química , Fosfopéptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteína Activadora de GTPasa p120/genética , Proteína Activadora de GTPasa p120/metabolismo , Dominios Homologos src
14.
Biochem Soc Trans ; 46(6): 1713-1720, 2018 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-30514771

RESUMEN

Pseudoenzymes generally lack detectable catalytic activity despite adopting the overall protein fold of their catalytically competent counterparts, indeed 'pseudo' family members seem to be incorporated in all enzyme classes. The small GTPase enzymes are important signaling proteins, and recent studies have identified many new family members with noncanonical residues within the catalytic cleft, termed pseudoGTPases. To illustrate recent discoveries in the field, we use the p190RhoGAP proteins as an example. p190RhoGAP proteins (ARHGAP5 and ARHGAP35) are the most abundant GTPase activating proteins for the Rho family of small GTPases. These are key regulators of Rho signaling in processes such as cell migration, adhesion and cytokinesis. Structural biology has complemented and guided biochemical analyses for these proteins and has allowed discovery of two cryptic pseudoGTPase domains, and the re-classification of a third, previously identified, GTPase-fold domain as a pseudoGTPase. The three domains within p190RhoGAP proteins illustrate the diversity of this rapidly expanding pseudoGTPase group.


Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Proteínas Activadoras de GTPasa/genética , Humanos , Unión Proteica , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/genética
15.
Structure ; 26(11): 1451-1461.e4, 2018 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-30174148

RESUMEN

The pseudoGTPases are a rapidly growing and important group of pseudoenzymes. p190RhoGAP proteins are critical regulators of Rho signaling and contain two previously identified pseudoGTPase domains. Here we report that p190RhoGAP proteins contain a third pseudoGTPase domain, termed N-GTPase. We find that GTP constitutively purifies with the N-GTPase domain, and a 2.8-Å crystal structure of p190RhoGAP-A co-purified with GTP reveals an unusual GTP-Mg2+ binding pocket. Six inserts in N-GTPase indicate perturbed catalytic activity and inability to bind to canonical GTPase activating proteins, guanine nucleotide exchange factors, and effector proteins. Biochemical analysis shows that N-GTPase does not detectably hydrolyze GTP, and exchanges nucleotide only under harsh Mg2+ chelation. Furthermore, mutational analysis shows that GTP and Mg2+ binding stabilizes the domain. Therefore, our results support that N-GTPase is a nucleotide binding, non-hydrolyzing, pseudoGTPase domain that may act as a protein-protein interaction domain. Thus, unique among known proteins, p190RhoGAPs contain three pseudoGTPase domains.


Asunto(s)
Guanosina Trifosfato/metabolismo , Magnesio/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Seudogenes , Ratas , Proteínas Represoras/genética
16.
Nat Commun ; 8(1): 506, 2017 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-28894085

RESUMEN

The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility. Here we report the discovery of two evolutionarily conserved GTPase-like domains located in the 'middle domain', previously thought to be unstructured. Deletion of these domains reduces RhoGAP activity. Crystal structures, MANT-GTPγS binding, thermal denaturation, biochemical assays and sequence homology analysis all strongly support defects in nucleotide-binding activity. Analysis of p190RhoGAP proteins therefore indicates the presence of two previously unidentified domains which represent an emerging group of pseudoenzymes, the pseudoGTPases.A growing number of 'pseudoenzymes' with a regulatory role in signal transduction processes but without catalytic activity are being identified. Here, the authors identify two pseudoGTPase domains in p190RhoGAP, characterize them biochemically and structurally and show that they influence RhoGAP activity.


Asunto(s)
Proteínas de Drosophila/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Represoras/metabolismo , Animales , Pollos , Cristalización , Proteínas de Drosophila/química , Drosophila melanogaster , GTP Fosfohidrolasas/química , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Poríferos , Dominios Proteicos , Pliegue de Proteína , Ratas , Proteínas Represoras/química , Homología de Secuencia
17.
Proc Natl Acad Sci U S A ; 113(43): 12238-12243, 2016 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-27791010

RESUMEN

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.


Asunto(s)
Histonas/genética , Neoplasias Ováricas/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias Uterinas/genética , Anciano , Anciano de 80 o más Años , Carcinosarcoma/genética , Carcinosarcoma/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Proteínas de Unión al ADN/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Mutación , Neoplasias Ováricas/patología , Fosfohidrolasa PTEN/genética , Telomerasa/genética , Neoplasias Uterinas/patología
18.
Hepatology ; 63(6): 1977-86, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26874653

RESUMEN

UNLABELLED: Despite advances in the diagnosis and management of idiopathic noncirrhotic portal hypertension, its pathogenesis remains elusive. Insight may be gained from study of early-onset familial idiopathic noncirrhotic portal hypertension, in which Mendelian mutations may account for disease. We performed exome sequencing of eight subjects from six kindreds with onset of portal hypertension of indeterminate etiology during infancy or childhood. Three subjects from two consanguineous families shared the identical rare homozygous p.N46S mutation in DGUOK, a deoxyguanosine kinase required for mitochondrial DNA replication; haplotype sharing demonstrated that the mutation in the two families was inherited from a remote common ancestor. All three affected subjects had stable portal hypertension with noncirrhotic liver disease for 6-16 years of follow-up. This mutation impairs adenosine triphosphate binding and reduces catalytic activity. Loss-of-function mutations in DGUOK have previously been implicated in cirrhosis and liver failure but not in isolated portal hypertension. Interestingly, treatment of patients with human immunodeficiency viral infection with the nucleoside analogue didanosine is known to cause portal hypertension in a subset of patients and lowers deoxyguanosine kinase levels in vitro; the current findings implicate these effects on deoxyguanosine kinase in the causal mechanism. CONCLUSION: Our findings provide new insight into the mechanisms mediating inherited and acquired noncirrhotic portal hypertension, expand the phenotypic spectrum of DGUOK deficiency, and provide a new genetic test for a specific cause of idiopathic noncirrhotic portal hypertension. (Hepatology 2016;63:1977-1986).


Asunto(s)
Hipertensión Portal/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Adolescente , Secuencia de Aminoácidos , Animales , Bovinos , Niño , Preescolar , Análisis Mutacional de ADN , Perros , Femenino , Genes Recesivos , Homocigoto , Humanos , Lactante , Fallo Hepático/genética , Masculino , Datos de Secuencia Molecular , Linaje , Análisis de Componente Principal , Ratas , Adulto Joven
19.
J Biol Chem ; 290(5): 2842-53, 2015 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-25525273

RESUMEN

Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 Å co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1(NPX(Y/F)3), revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1(NPX(Y/F)3). We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Portadoras/genética , Cromatografía en Gel , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Humanos , Inmunoprecipitación , Proteína KRIT1 , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Mutación Missense , Mutación Puntual/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética
20.
Nat Genet ; 46(10): 1135-1139, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25217960

RESUMEN

Upon detection of pathogen-associated molecular patterns, innate immune receptors initiate inflammatory responses. These receptors include cytoplasmic NOD-like receptors (NLRs) whose stimulation recruits and proteolytically activates caspase-1 within the inflammasome, a multiprotein complex. Caspase-1 mediates the production of interleukin-1 family cytokines (IL1FCs), leading to fever and inflammatory cell death (pyroptosis). Mutations that constitutively activate these pathways underlie several autoinflammatory diseases with diverse clinical features. We describe a family with a previously unreported syndrome featuring neonatal-onset enterocolitis, periodic fever, and fatal or near-fatal episodes of autoinflammation. We show that the disease is caused by a de novo gain-of-function mutation in NLRC4 encoding a p.Val341Ala substitution in the HD1 domain of the protein that cosegregates with disease. Mutant NLRC4 causes constitutive IL1FC production and macrophage cell death. Infected macrophages from affected individuals are polarized toward pyroptosis and exhibit abnormal staining for inflammasome components. These findings identify and describe the cause of a life-threatening but treatable autoinflammatory disease that underscores the divergent roles of the NLRC4 inflammasome.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/genética , Proteínas de Unión al Calcio/genética , Enterocolitis/genética , Inflamación/genética , Mutación Missense , Exoma/genética , Salud de la Familia , Resultado Fatal , Femenino , Humanos , Masculino , Linaje , Análisis de Secuencia de ADN , Síndrome
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