Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot

Self-assembling protein nanoparticles in the design of vaccines: 2022 update

Vaccines constitute a pillar in the prevention of infectious diseases. The unprecedented emergence of novel immunization strategies due to the COVID-19 pandemic has again positioned vaccination as a pivotal measure to protect humankind and reduce the clinical impact and socioeconomic burden worldwide. Vaccination pursues the ultimate goal of eliciting a protective response in immunized individuals. To achieve this, immunogens must be efficiently delivered to prime the immune system and produce robust protection. Given their safety, immunogenicity, and flexibility to display varied and native epitopes, self-assembling protein nanoparticles represent one of the most promising immunogen delivery platforms. Currently marketed vaccines against the human papillomavirus, for instance, illustrate the potential of these nanoassemblies. This review is intended to provide novelties, since 2015, on the ground of vaccine design and self-assembling protein nanoparticles, as well as a comparison with the current emergence of mRNA-based vaccines. © 2022 by the authors.Jacinto López Sagaseta is a Ramón y Cajal Investigator, grant RYC-2017-21683, Ministry of Science and Innnovation of Spain. Nerea Ugidos-Damboriena is a recipient of a María Zambrano contract funded by UPNA and the Ministry of Universities of Spain within the Plan of Recovery, Transformation and Resilience and the European Recovery Instrument Next Generation EU

Functional dissection of inherited non-coding variation influencing multiple myeloma risk

Funding Information: This work was supported by grants from the Knut and Alice Wallenberg Foundation (2012.0193 and 2017.0436), the Swedish Research Council (2017-02023 and 2018-00424), the Swedish Cancer Society (2017/265), the Nordic Cancer Union (R217-A13329-18-S65), Arne and Inga-Britt Lundberg’s Stiftelse (2017-0055), European Research Council (EU-MSCA-COFUND 754299 CanFaster), Myeloma UK and Cancer Research UK (C1298/A8362), The National Institute of Health (R01 DK103794 and R01HL146500), the New York Stem Cell Foundation, a gift from the Lodish Family to Boston Children’s Hospital, and Mr. Ralph Stockwell. We thank Ellinor Johnsson for her assistance between 2011 and 2020. We are indebted to the patients who participated in the study. Publisher Copyright: © 2022, The Author(s).Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.Peer reviewe

Interactome of the Autoimmune Risk Protein ANKRD55

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4(+) T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, beta-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.This work was supported by the following grants to KV: Grupos de Investigacion (IT512-10, PPG17/44) and MINECO (SAF2016-74891R). NU is recipient of a predoctoral studentship from the Gobierno Vasco (Reference PRE-2013-1-891). CIC bioGUNE is accredited with the Severo Ochoa Excellence award by the Spanish Ministerio de Econom i a y Competitividad, MINECO (SEV-2016-0644)

Identification of regulators of hematopoietic stem and progenitor cells in vivo in humans using population genetics

Introduction: Understanding how hematopoietic stem- and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and for regenerative medicine. Traditionally, however, HSPC regulation has been studied in model systems, and little is known about the situation in vivo in humans. Methods: To learn how HSPCs are regulated under native conditions in humans, we carried out a first large-scale genome-wide association study on CD34+ cells, representing HSPCs in blood. We used circulating CD34+ levels as a proxy trait to expose regulators of key phenomena like HSPC pool size, migration, and early differentiation. We created a unique phenotyping platform based on high-throughput, high-resolution flow-cytometry and machine learning-based algorithms for automated flow data analysis, and quantified CD34+ cells in 9,936 adults.Results: We identified 8 genome-wide (P<5x10-8) and 20 suggestive loci (P<5x10-6) associated with CD34+ levels. The two strongest were the HSPC migration receptor CXCR4 and a novel protein phosphatase never previously implicated in stem cell biology. Using eQTL, ATAC-seq, and promoter capture Hi-C analysis in isolated HSPCs, we pinpoint likely causal variants, including variants in distant regulatory elements selectively active in specific HSPC subpopulations. Furthermore, shRNA knockdown in primary CD34+ cells supports that some of the identified genes affect CD34+ proliferation and differentiation.Conclusions: We report the first large-scale analysis of the genetic architecture of HSPC regulation, with potential implications for stem cell transplantation and the treatment of hematologic malignancies.Grant information: European Research Council, Swedish Research Council, Swedish Cancer Society

The potential impact of plant biotechnology on the Millennium Development Goals

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world's poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today's political climate depends on how we deal with current barriers to adoption

Genome-wide association study on 13 167 individuals identifies regulators of blood CD34+cell levels

Stem cell transplantation is a cornerstone in the treatment of blood malignancies. The most common method to harvest stem cells for transplantation is by leukapheresis, requiring mobilization of CD34+ hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the blood. Identifying the genetic factors that control blood CD34+ cell levels could reveal new drug targets for HSPC mobilization. Here we report the first large-scale, genome-wide association study on blood CD34+ cell levels. Across 13 167 individuals, we identify 9 significant and 2 suggestive associations, accounted for by 8 loci (PPM1H, CXCR4, ENO1-RERE, ITGA9, ARHGAP45, CEBPA, TERT, and MYC). Notably, 4 of the identified associations map to CXCR4, showing that bona fide regulators of blood CD34+ cell levels can be identified through genetic variation. Further, the most significant association maps to PPM1H, encoding a serine/threonine phosphatase never previously implicated in HSPC biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. Through functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates an MYB transcription factor–binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, PPM1H knockdown increases the proportion of CD34+ and CD34+90+ cells in cord blood assays. Our results provide the first large-scale analysis of the genetic architecture of blood CD34+ cell levels and warrant further investigation of PPM1H as a potential inhibition target for stem cell mobilization

Genome-wide association study on 13,167 individuals identifies regulators of hematopoietic stem and progenitor cell levels in human blood

Understanding how hematopoietic stem and progenitor cells (HSPCs) are regulated is of central importance for the development of new therapies for blood disorders and stem cell transplantation. To date, HSPC regulation has been extensively studied in vitro and in animal models, but less is known about the mechanisms in vivo in humans. Here, in a genome-wide association study on 13,167 individuals, we identify 9 significant and 2 suggestive DNA sequence variants that influence HSPC (CD34+) levels in human blood. The identified loci associate with blood disorders, harbor known and novel HSPC genes, and affect gene expression in HSPCs. Interestingly, our strongest association maps to the PPM1H gene, encoding an evolutionarily conserved serine/threonine phosphatase never previously implicated in stem cell biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. By functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates a MYB transcription factor binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, rs772557-A selectively increases HSPC subpopulations in which the MYB site is active, and PPM1H shRNA- knockdown increased CD34+ and CD34+90+ cell proportions in umbilical cord blood cultures. Our findings represent the first large-scale association study on a stem cell trait, illuminating HSPC regulation in vivo in humans, and identifying PPM1H as a novel inhibition target that can potentially be utilized clinically to facilitate stem cell harvesting for transplantation