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

MEPSAnd: Minimum Energy Path Surface Analysis over n-dimensional surfaces.

Summary: Understanding biophysical phenomena from the approach of molecular simulation is becoming the state-of-art in many research and technology development fields. Energy surfaces with more than 3 dimensions (2 coordinates and energy) are now computationally accessible, yet interpreting the information they offer is not straightforward and the tasks involved very time-consuming. Here we present MEPSAnd, an open source GUI-based program that natively calculates minimum energy paths across energy surfaces of an arbitrary number of dimensions. In addition to the multidimensional analysis of path through lowest barriers, MEPSAnd can also automatically calculate a finite series of suboptimal paths. To allow the efficient interpretation of results, MEPSAnd offers three distinct plotting solutions: i) energy profiles, ii) coordinate projections and iii) network projections. GUI-independent pipelines are also supported via direct python scripting. Therefore, MEPSAnd is a powerful user friendly tool that streamlines path-finding tasks on n-dimensional energy surfaces.pre-print1996 K

De novo heterozygous mutations in SMC3 cause a range of cornelia de lange syndrome-overlapping phenotypes

© 2015 WILEY PERIODICALS, INC. 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 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with 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. Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder caused by mutation in five genes encoding subunits or regulators of the cohesin complex. To date, only the clinical features of the unique mildly affected CdLS male with SMC3 mutation have been published. Here, we report a series of 16 probands with 15 different intragenic mutations in SMC3 that provide a significant advance in our understanding of the clinical and molecular basis of Cornelia de Lange syndrome and overlapping phenotypes.CdLS Foundation of UK and Ireland for their long-term help and support. M.A.D. and I.D.K. are indebted to the USA Cornelia de Lange Syndrome FoundationPeer Reviewe

MEPSA: Minimum energy pathway analysis for energy landscapes

From conformational studies to atomistic descriptions of enzymatic reactions, potential and free energy landscapes can be used to describe biomolecular systems in detail. However, extracting the relevant data of complex 3D energy surfaces can sometimes be laborious. In this article, we present MEPSA (Minimum Energy Path Surface Analysis), a cross-platform user friendly tool for the analysis of energy landscapes from a transition state theory perspective. Some of its most relevant features are: identification of all the barriers and minima of the landscape at once, description of maxima edge profiles, detection of the lowest energy path connecting two minima and generation of transition state theory diagrams along these paths. In addition to a built-in plotting system, MEPSA can save most of the generated data into easily parseable text files, allowing more versatile uses of MEPSA's output such as the generation of molecular dynamics restraints from a calculated path.Grant IPT2011-0964-900000 (Government of Spain).Peer Reviewe

Fisioterapia Gineco-Obstétrica en la prevención de traumatismo perineal en el parto y disfunciones del suelo pélvico postparto

INTRODUCCIÓN: La disfunción del suelo pélvico está ligada al embarazo y parto por hipersolicitación mecánica de los tejidos músculo-conjuntivos y nerviosos. Es frecuente el sufrimiento de traumatismo perineal en el parto. Por lo tanto, deben ser objeto de intervención en materia de prevención desde el campo de la fisioterapia gineco-obstétrica. OBJETIVO: Estudiar la evidencia científica existente sobre la intervención de la fisioterapia gineco-obstétrica en la prevención del traumatismo perineal y de disfunciones del suelo pélvico postparto. MÉTODOS: La búsqueda se realizó en Pubmed, Science Direct, Dialnet, PEDro y Alcorze. Los estudios seleccionados debían ser revisiones sistemáticas, estudios aleatorios controlados y meta-análisis, publicados entre 2005 y 2015, que tratasen la prevención del traumatismo perineal y disfunción del suelo pélvico, cuya calidad metodológica se valoró a través de la escala PEDro. Finalmente, se incluyeron 12 artículos para analizar. RESULTADOS: El masaje perineal realizado en la segunda fase del parto aumenta la probabilidad de mantener la integridad perineal; mientras que en el periodo de gestación, no posee efecto protector ni perjudicial. El trabajo con el dispositivo EPI-NO® entre la semana 37 de gestación y el parto reduce el riesgo de traumatismo perineal. Y el entrenamiento de la musculatura del suelo pélvico (EMSP) muestra menor incidencia de incontinencia urinaria, tanto en el embarazo como en el postparto, por aumento de la fuerza de la musculatura y de la presión intrauretral, pero requiere su aprendizaje específico. CONCLUSIÓN: La aplicación de fisioterapia gineco-obstétrica muestra un efecto protector del periné, reduciéndose la incidencia de disfunción del suelo pélvico. Es necesaria la concienciación y motivación de su prevención, así como mayor investigación

A novel RAD21 p.(Gln592del) variant expands the clinical description of Cornelia de Lange syndrome type 4 – Review of the literature.

Cornelia de Lange syndrome (CdLS) is a heterogeneous developmental disorder where 70% of clinically diagnosed patients harbor a mutation in one of five CdLS associated cohesin proteins. Around 500 mutations have been identified to cause CdLS, however only eight different alterations are identified in RAD21, encoding the RAD21 cohesin protein that constitute the link between SMC1A and SMC3 within the cohesin ring. We report a 15- month-old boy presenting with developmental delay, distinct CdLS facial features, gastrointestinal reflux in early infancy, testis retention fetal pads and diaphragmatic hernia. Exome sequencing revealed a novel RAD21 variant, c.1774_1776del; p.(Gln592del), suggestive of CdLS type 4. Segregation analysis of the two healthy parents confirmed the variant as de novo and bioinformatic analysis predicted the variant as disease-causing. Functional assessment by in silico structural model predicted that the p.Gln592del variant results in a discontinued contact between RAD21-Lys591 and the SMC1A residues Glu1191 and Glu1192, causing changes in the RAD21-SMC1A interface. In conclusion, we report a novel RAD21 p.(Glu592del) variant that expands the clinical description of CdLS type 4 and validate the pathogenicity of the variant by in silico structural modeling that displayed disturbed RAD21-SMC1A interface.pre-print2,82 M

Human Mitochondrial HMG-CoA Synthase Deficiency: Role of Enzyme Dimerization Surface and Characterization of Three New Patients.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mitochondrial HMG-CoA synthase deficiency or mHS deficiency, OMIM #605911) is an inborn error of metabolism that affects ketone body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycemia and dicarboxylic aciduria. The diagnosis is difficult due to the relatively unspecific clinical and biochemical presentation, and fewer than 30 patients have been described. This work describes three new patients with mHS deficiency and two missense mutations c.334C>T (p.R112W) and c.430G>T (p.V144L) previously not reported. We developed a new method to express and measure the activity of the enzyme and in this work the study is extended to ten new missense variants including those of our patients. Enzymatic assays showed that three of the mutant proteins retained some but seven completely lacked activity. The identification of a patient homozygous for a mutation that retains 70% of enzyme activity opens the door to a new interpretation of the disease by demonstrating that a modest impairment of enzyme function can actually produce symptoms. This is also the first study employing molecular dynamics modelling of the enzyme mutations. We show that the correct maintenance of the dimerization surface is crucial for retaining the structure of the active center and therefore the activity of the enzyme.post-print1746 K

Pathogenic convergence of CNVs in genes functionally associated to a severe neuromotor developmental delay syndrome.

Background Complex developmental encephalopathy syndromes might be the consequence of unknown genetic alterations that are likely to contribute to the full neurological phenotype as a consequence of pathogenic gene combinations. Methods To identify the additional genetic contribution to the neurological phenotype, we studied as a test case a boy, with a KCNQ2 exon-7 partial duplication, by single-nucleotide polymorphism (SNP) microarray to detect copy-number variations (CNVs). Results The proband presented a cerebral palsy like syndrome with a severe motor and developmental encephalopathy. The SNP array analysis detected in the proband several de novo CNVs, nine partial gene losses (LRRC55, PCDH9, NALCN, RYR3, ELAVL2, CDH13, ATP1A2, SLC17A5, ANO3), and two partial gene duplications (PCDH19, EFNA5). The biological functions of these genes are associated with ion channels such as calcium, chloride, sodium, and potassium with several membrane proteins implicated in neural cell-cell interactions, synaptic transmission, and axon guidance. Pathogenically, these functions can be associated to cerebral palsy, seizures, dystonia, epileptic crisis, and motor neuron dysfunction, all present in the patient. Conclusions Severe motor and developmental encephalopathy syndromes of unknown origin can be the result of a phenotypic convergence by combination of several genetic alterations in genes whose physiological function contributes to the neurological pathogenic mechanism.post-print783 K

Lacasa de alto potencial redox funcional en sangre mediante evolución dirigida método de obtención y sus aplicaciones

La presente invención describe una lacasa de alto potencial redox obtenida mediante evolución molecular dirigida que es activa en condiciones electrofisiológicas, que resiste elevadas concentraciones de haluros, que tiene una actividad significativa a pHs neutros/alcalinos y que es activa en sangre y plasma humano. La presente invención se refiere a la secuencia aminoacídica de dicha lacasa, a la secuencia nucleotídica que codifica para dicha lacasa y células que permiten su obtención. La lacasa de la invención presenta aplicaciones en diversos sectores: nano-biotecnología, biomedicina, procesos de biorremediación, industria papelera y química fina.Peer reviewedConsejop Superior de Investigaciones CientíficasB1 Patente sin examen previ

Lacasa de alto potencial redox funcional en sangre mediante evolución dirigida método de obtención y sus aplicaciones

La presente invención describe una lacasa de alto potencial redox obtenida mediante evolución molecular dirigida que es activa en condiciones electrofisiológicas, que resiste elevadas concentraciones de haluros, que tiene una actividad significativa a pHs neutros/alcalinos y que es activa en sangre y plasma humano. La presente invención se refiere a la secuencia aminoacídica de dicha lacasa, a la secuencia nucleotídica que codifica para dicha lacasa y células que permiten su obtención. La lacasa de la invención presenta aplicaciones en diversos sectores: nano-biotecnología, biomedicina, procesos de biorremediación, industria papelera y química fina.Peer reviewedConsejop Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic