Structural basis for the inactivation of cytosolic DNA sensing by the vaccinia virus

Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 – Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.his work was funded by “la Caixa” Foundation, Health Research grant 2020 (LCF/PR/HR20/52400017) to MP and OL, by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI) grants SAF2015-64869-R-FEDER and RTI2018-094211-B-100-FEDER to MP, and SAF2017-82632-P to OL, co-funded by the European Regional Development Fund (ERDF); the support of Catalan Government (grant 2017 SGR 961) to MP, and the support of the National Institute of Health Carlos III to CNIO; grants 31 Y2018/BIO4747 and P2018/NMT4443 from the Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund to OL. CFR is funded by BES-2015-071348 PhD fellowship by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI). We gratefully acknowledge institutional funding from the Spanish State Research Agency of the Spanish Ministry of Science and Innovation – Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia -Centres of Excellence “Severo Ochoa” CEX2019-000891-S and CEX2019-000913-S. IRB Barcelona is a member of the CERCA System of the Catalan Government P.B. is supported by a CIBERER contract.Peer ReviewedPostprint (author's final draft

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies

Análisis de la inserción laboral de personas con discapacidad iIntelectual: La experiencia de la Universidad de Valladolid

Producción CientíficaAnálisis de la inserción laboral de personas con discapacidad intelectual

Estructura y determinantes funcionales de selectividad de sustrato de los transportadores HAT para aminoácidos neutros

[spa] Los transportadores heteroméricos de aminoácidos (HAT) se encuentran compuestos por una subunidad pesada que dirige el tráfico del transportador a la membrana plasmática y la subunidad ligera que posee la actividad catalítica. Cada uno de sus miembros ha evolucionado para reconocer y transportar una serie de aminoácidos específicos, lo cual les confiere un papel fisiológico cuya alteración se ha visto relacionada con diferentes patologías humanas; cáncer, aminoacidurias, sordera asociada a la edad o autismo. De la aplicación de inhibidores que reconozcan estos transportadores de manera específica y de la comprensión de cómo una mutación afecta a su actividad radica la importancia de caracterizar estructural y funcionalmente estos transportadores. Hasta hace muy pocos años la información estructural publicada de los HATs se reducía a modelos atómicos construidos a partir de homólogos bacterianos lejanos y una estructura de h4F2hc/LAT2 resuelta a 21 Å. No obstante, en los 2 últimos años, se han resuelto varias estructuras de un LAT bacteriano y de hasta tres HATs diferentes, incluyendo uno en varias conformaciones, en presencia y ausencia de sustrato. Durante esta tesis se ha realizado un cribado de varios HATs, con el objetivo de encontrar aquel que mejor balance presentara entre un alto nivel de expresión y purificación, y una homogeneidad y estabilidad suficientes para realizar un estudio estructural en crio-microscopía electrónica. Tal estudio resultó en la resolución de la estructura de 4F2hc/LAT2 humano a una resolución de 3,7 Å con el núcleo transmembranal de LAT2 resuelto en un rango entre 2,5 y 3 Å. El análisis de la estructura de 4F2hc/LAT2, junto con una aproximación multidisciplinar, ha permitido establecer las bases del diferente perfil de selectividad mostrado por los transportadores HAT de aminoácidos neutros (LAT2, LAT1 y Asc1). De este modo, la información estructural existente (LAT1 y LAT2 humanos) junto con los estudios funcionales llevados a cabo para LAT2 y Asc1 humanos y los análisis de simulación con sustratos realizados en LAT2 humano permitieron identificar los residuos clave para la diferente selectividad de sustrato dentro de los transportadores HAT de aminoácidos neutros. Por otro lado, el análisis estructural de las mutaciones identificadas en LAT2 asociadas a sordera temprana y cataratas en pacientes determinaron que la región entre el dominio transmembrana 2 (TM2) y el bucle intercelular 1 (IL1) resultaría clave en la configuración del sitio de unión al sustrato. De esta manera, proponemos que las conexiones de regiones cercanas al sitio de unión con el TM2 y el IL1 resultarían clave en la conformación espacial de los residuos clave para el reconocimiento de sustrato y que han sido identificados en el transcurso de esta tesis. Así, pequeños cambios en las interacciones entre estas regiones que se observan en el análisis de secuencia de los diferentes HATs podrían explicar, al menos en parte, el perfil de selectividad de sustrato observados para los diferentes subgrupos de transportadores HAT. Nuestros resultados abren la puerta para identificar los mecanismos moleculares de selectividad de sustratos de los transportadores HAT tanto en el lugar de unión al sustrato como en una región que determina la conformación de este lugar de unión

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field