Computational modeling of cohesin ATPase dynamics

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física Teórica de la Materia Condensada. Fecha de lectura: 20-04-2018El complejo de cohesina humano participa en procesos de segregación cromosómica, reparación de ADN, organización de cromatina y regulación de la transcripción. Este complejo, que forma un anillo que atrapa topológicamente DNA, está formado por cuatro subunidades principales: Smc1A, Smc3, Rad21 y Stag1/2. Determinadas variantes de componentes de este complejo que se han relacionado con cohesinopatías y cáncer. En esta tesis se han empleado una serie de métodos de biología estructural computacional (modelado por homología, técnicas de dinámica molecular clásica, dirigida y de potenciales híbridos de mecánica cuántica y mecánica clásica, así como docking molecular) para elucidar aspectos relevantes de la dinámica de cohesinas vinculada a la actividad ATPasa. Además, se ha desarrollado MEPSA, una herramienta accesible que permite estandarizar el análisis de super cies de energía libre tridimensionales (un tipo de dato que resulta frecuente en múltiples protocolos de dinámica molecular). Los resultados obtenidos en esta tesis han permitido: i) describir cómo la unión del dominio C-terminal de Rad21 a la cabeza de Smc1A facilita la actividad ATPasa en el centro activo de Smc1A en el anillo de cohesina, ii) caracterizar que esta actividad ATPasa desencadena un proceso de acoplamiento alostérico entre los centros activos de Smc1A y Smc3 no descrito previamente y iii) evaluar el efecto desestabilizador que tiene la hidrólisis de ATP en ambos centros activos sobre la interfaz formada por los dominios cabeza de Scm1A y Smc3. Asimismo, la descripción a escala atómica que ofrecen los modelos generados ha permitido racionalizar múltiples variantes relacionadas con enfermedades humanas (síndrome de Cornelia de Lange y cáncer) y mutaciones no neutras en levadura, así como la detección de una nueva diana molecular para la búsqueda computacional de fármacos. Los resultados preliminares de un protocolo de docking molecular contra dicha diana han permitido identi car una molécula capaz de inducir arresto en fase G2/M del ciclo celular en la línea celular humana 293T.Human cohesin complex is involved in processes of chromosome segregation, DNA repair, chromatin organization and transcription regulation. This complex, which forms a ring capable of topologically entrapping DNA, is formed by four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. Variants a ecting members of this complex have been directly related to cohesinopathies and cancer. In this thesis a series of computational structural biology methods (homology modeling, classical, biased and hybrid quantum mechanics/molecular mechanics molecular dynamics techniques, as well as molecular docking) have been used to elucidate relevant aspects of cohesin ATPase dynamics. In addition, a user-friendly free energy analysis software (MEPSA) has been developed, streamlining the analysis of 3D free energy surfaces (a common type of data generated in many molecular dynamics protocols). The results presented in this thesis have allowed to: i) describe how Rad21 C-terminal domain binding to the Smc1A head domain facilitates ATPase activity in the Smc1A active site of a cohesin ring, ii) characterize that this ATPase activity triggers a previously unreported allosteric coupling mechanism between Smc1A and Smc3 active sites and iii) quantify the destabilizing e ect ATP hydrolysis in both active sites has over the interface formed by Smc1A and Smc3 head domains. This structural framework has made it possible to rationalize many disease-related (Cornelia de Lange syndrome and cancer) human variants and yeast non-neutral mutations, also revealing a new molecular target for in silico drug discovery. Preliminary results of a molecular docking protocol against this target have yielded a drug capable of inducing G2/M cell cycle arrest in the 293T human cell line

Evaluating Face2Gene as a Tool to Identify Cornelia de Lange Syndrome by Facial Phenotypes

Characteristic or classic phenotype of Cornelia de Lange syndrome (CdLS) is associated with a recognisable facial pattern. However, the heterogeneity in causal genes and the presence of overlapping syndromes have made it increasingly difficult to diagnose only by clinical features. DeepGestalt technology, and its app Face2Gene, is having a growing impact on the diagnosis and management of genetic diseases by analysing the features of affected individuals. Here, we performed a phenotypic study on a cohort of 49 individuals harbouring causative variants in known CdLS genes in order to evaluate Face2Gene utility and sensitivity in the clinical diagnosis of CdLS. Based on the profile images of patients, a diagnosis of CdLS was within the top five predicted syndromes for 97.9% of our cases and even listed as first prediction for 83.7%. The age of patients did not seem to affect the prediction accuracy, whereas our results indicate a correlation between the clinical score and affected genes. Furthermore, each gene presents a different pattern recognition that may be used to develop new neural networks with the goal of separating different genetic subtypes in CdLS. Overall, we conclude that computer-assisted image analysis based on deep learning could support the clinical diagnosis of CdL

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder

International audienceDe novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity

De novo Heterozygous Mutations in SMC3 Cause a Range of Cornelia De Lange Syndrome-Overlapping Phenotypes

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of sixteen patients with CdLS-like features caused by mutations in SMC3. Modelling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared to typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ¡­1-2% of CdLS-like phenotypes.Fil: Gil Rodríguez, María Concepción. Universidad de Zaragoza; EspañaFil: Deardorff, Matthew A.. Children´s Hospital of Philadelphia; Estados Unidos. University of Pennsylvania ; Estados UnidosFil: Ansari, Morad. University of Edinburgh; Reino UnidoFil: Tan, Christopher A.. University of Chicago; Estados UnidosFil: Parenti, Ilaria. Universität zu Lübeck; Alemania. Università degli Studi di Milano; ItaliaFil: Baquero Montoya, Carolina. Universidad de Zaragoza; España. Hospital Pablo Tobón Uribe; ColombiaFil: Ousager, Liliana B.. Odense University Hospital; DinamarcaFil: Puisac, Beatriz. Universidad de Zaragoza; EspañaFil: Hernández Marcos, María. Universidad de Zaragoza; EspañaFil: Teresa Rodrigo, María Esperanza. Universidad de Zaragoza; EspañaFil: Marcos Alcalde, Iñigo. Centro de Biología Molecular ; EspañaFil: Wesselink, Jan-Jaap. Biomol‐Informatics SL; EspañaFil: Lusa Bernal, Silvia. Biomol‐Informatics SL; EspañaFil: Bijlsma, Emilia K.. Leiden University; Países BajosFil: Braunholz, Diana. Universität zu Lübeck; AlemaniaFil: Bueno Martínez, Inés. Universidad de Zaragoza; España. Hospital Clínico Universitario ; EspañaFil: Clark, Dina. Children´s Hospital of Philadelphia; Estados UnidosFil: Cooper, Nicola S.. Birmingham Women´s Hospital; Reino UnidoFil: Curry, Cynthia J.. University of California; Estados UnidosFil: Fisher, Richard. The James Cook University Hospital; Reino UnidoFil: Fryer, Alan. Liverpool Women´s Hospital and Alder Hey Children´s Hospital; Reino UnidoFil: Ganesh, Jaya. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Gervasini, Cristina. Università degli Studi di Milano; ItaliaFil: Gillessen Kaesbach, Gabrielle. Universität zu Lübeck; AlemaniaFil: Guo, Yiran. Children´s Hospital of Philadelphia; Estados UnidosFil: Hakonarson, Hakon. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Hopkin, Robert J.. Cincinnati Children´s Hospital Medical Center; Estados UnidosFil: Kaur, Maninder. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Keating, Brendan J.. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Kibaek, María. HC Andersen Children´s Hospital; DinamarcaFil: Kinning, Esther. Southern General Hospital; Reino UnidoFil: Kleefstra, Tjitske. Radboud Universiteit Nijmegen; Países BajosFil: Kline, Antonie D.. Greater Baltimore Medical Center; Estados UnidosFil: Kuchinskaya, Ekaterina. Linköping University Hospital; SueciaFil: Larizza, Lidia. Università degli Studi di Milano; ItaliaFil: Li, Yun R.. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Liu, Xuanzhu. BGI-Shenzhen; ChinaFil: Mariani, Milena. Fobdazione MBBM AOS Gerardo; ItaliaFil: Picker, Jonathan D.. Boston Children´s Hospital; Estados UnidosFil: Pié, Ángeles. Universidad de Zaragoza; EspañaFil: Pozojevic, Jelena. Universität zu Lübeck; AlemaniaFil: Queralt, Ethel. L´Hospitalet de Llobregat; EspañaFil: Richer, Julie. University of Ottawa; CanadáFil: Roeder, Elizabeth. University of Texas San Antonio; Estados UnidosFil: Sinha, Anubha. Birmingham Women´s Hospital; Reino UnidoFil: Scott, Richard H.. Great Ormond Street Hospital; Reino Unido. UCL Intitute of Child Health; Reino UnidoFil: So, Joyce. CAMH; CanadáFil: Wusik, Katherine A.. Cincinnati Children´s Hospital Medical Center; Estados UnidosFil: Wilson, Louise. Great Ormond Street Hospital; Reino UnidoFil: Zhang, Jianguo. BGI-Shenzhen; ChinaFil: Gómez Puertas, Paulino. Centro de Biología Molecular ; EspañaFil: Casale, Cesar Horacio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ström, Lena. Karolinska Institutet; SueciaFil: Selicorni, Angelo. Fobdazione MBBM AOS Gerardo; ItaliaFil: Ramos, Feliciano J.. Universidad de Zaragoza; España. Hospital Clínico Universitario ; EspañaFil: Jackson. Laird G.. Drexel University; Estados UnidosFil: Krantz, Ian D.. University of Pennsylvania ; Estados Unidos. Children´s Hospital of Philadelphia; Estados UnidosFil: Das, Soma. University of Chicago; Estados UnidosFil: Hennekam, Raoul C.M.. University of Amsterdam; Países BajosFil: Kaiser, Frank J.. Universität zu Lübeck; AlemaniaFil: FitzPatrick, David R.. University of Edinburgh; Reino UnidoFil: Pié, Juan. Universidad de Zaragoza; Españ