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1.
Cell ; 187(12): 2919-2934.e20, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38761800

RESUMEN

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.


Asunto(s)
Vacunas contra el SIDA , Anticuerpos Neutralizantes , Linfocitos B , Anticuerpos Anti-VIH , VIH-1 , Humanos , Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Linaje de la Célula , Liposomas , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Mutación , Proteína gp41 de Envoltorio del VIH/inmunología
2.
Cell ; 186(1): 63-79.e21, 2023 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-36608659

RESUMEN

Metabolism is deeply intertwined with aging. Effects of metabolic interventions on aging have been explained with intracellular metabolism, growth control, and signaling. Studying chronological aging in yeast, we reveal a so far overlooked metabolic property that influences aging via the exchange of metabolites. We observed that metabolites exported by young cells are re-imported by chronologically aging cells, resulting in cross-generational metabolic interactions. Then, we used self-establishing metabolically cooperating communities (SeMeCo) as a tool to increase metabolite exchange and observed significant lifespan extensions. The longevity of the SeMeCo was attributable to metabolic reconfigurations in methionine consumer cells. These obtained a more glycolytic metabolism and increased the export of protective metabolites that in turn extended the lifespan of cells that supplied them with methionine. Our results establish metabolite exchange interactions as a determinant of cellular aging and show that metabolically cooperating cells can shape the metabolic environment to extend their lifespan.


Asunto(s)
Longevidad , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Metionina/metabolismo , Transducción de Señal
3.
Cell ; 186(21): 4514-4527.e14, 2023 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-37757828

RESUMEN

Autozygosity is associated with rare Mendelian disorders and clinically relevant quantitative traits. We investigated associations between the fraction of the genome in runs of homozygosity (FROH) and common diseases in Genes & Health (n = 23,978 British South Asians), UK Biobank (n = 397,184), and 23andMe. We show that restricting analysis to offspring of first cousins is an effective way of reducing confounding due to social/environmental correlates of FROH. Within this group in G&H+UK Biobank, we found experiment-wide significant associations between FROH and twelve common diseases. We replicated associations with type 2 diabetes (T2D) and post-traumatic stress disorder via within-sibling analysis in 23andMe (median n = 480,282). We estimated that autozygosity due to consanguinity accounts for 5%-18% of T2D cases among British Pakistanis. Our work highlights the possibility of widespread non-additive genetic effects on common diseases and has important implications for global populations with high rates of consanguinity.


Asunto(s)
Consanguinidad , Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/genética , Homocigoto , Fenotipo , Polimorfismo de Nucleótido Simple , Bancos de Muestras Biológicas , Genoma Humano , Predisposición Genética a la Enfermedad , Reino Unido
4.
Cell ; 185(16): 3041-3055.e25, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35917817

RESUMEN

Rare copy-number variants (rCNVs) include deletions and duplications that occur infrequently in the global human population and can confer substantial risk for disease. In this study, we aimed to quantify the properties of haploinsufficiency (i.e., deletion intolerance) and triplosensitivity (i.e., duplication intolerance) throughout the human genome. We harmonized and meta-analyzed rCNVs from nearly one million individuals to construct a genome-wide catalog of dosage sensitivity across 54 disorders, which defined 163 dosage sensitive segments associated with at least one disorder. These segments were typically gene dense and often harbored dominant dosage sensitive driver genes, which we were able to prioritize using statistical fine-mapping. Finally, we designed an ensemble machine-learning model to predict probabilities of dosage sensitivity (pHaplo & pTriplo) for all autosomal genes, which identified 2,987 haploinsufficient and 1,559 triplosensitive genes, including 648 that were uniquely triplosensitive. This dosage sensitivity resource will provide broad utility for human disease research and clinical genetics.


Asunto(s)
Variaciones en el Número de Copia de ADN , Genoma Humano , Variaciones en el Número de Copia de ADN/genética , Dosificación de Gen , Haploinsuficiencia/genética , Humanos
5.
Annu Rev Biochem ; 90: 137-164, 2021 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-33556282

RESUMEN

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , Enzimas/metabolismo , Complejos Multiproteicos/genética , Animales , Enzimas/genética , Inestabilidad Genómica , Humanos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Recombinación V(D)J
6.
Nat Immunol ; 25(6): 957-968, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38811815

RESUMEN

The adult central nervous system (CNS) possesses a limited capacity for self-repair. Severed CNS axons typically fail to regrow. There is an unmet need for treatments designed to enhance neuronal viability, facilitate axon regeneration and ultimately restore lost neurological functions to individuals affected by traumatic CNS injury, multiple sclerosis, stroke and other neurological disorders. Here we demonstrate that both mouse and human bone marrow neutrophils, when polarized with a combination of recombinant interleukin-4 (IL-4) and granulocyte colony-stimulating factor (G-CSF), upregulate alternative activation markers and produce an array of growth factors, thereby gaining the capacity to promote neurite outgrowth. Moreover, adoptive transfer of IL-4/G-CSF-polarized bone marrow neutrophils into experimental models of CNS injury triggered substantial axon regeneration within the optic nerve and spinal cord. These findings have far-reaching implications for the future development of autologous myeloid cell-based therapies that may bring us closer to effective solutions for reversing CNS damage.


Asunto(s)
Axones , Factor Estimulante de Colonias de Granulocitos , Interleucina-4 , Ratones Endogámicos C57BL , Regeneración Nerviosa , Neutrófilos , Animales , Neutrófilos/inmunología , Regeneración Nerviosa/inmunología , Ratones , Humanos , Axones/metabolismo , Axones/fisiología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Interleucina-4/metabolismo , Activación Neutrófila , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/metabolismo , Traslado Adoptivo , Citocinas/metabolismo , Células Cultivadas
7.
Nat Immunol ; 25(7): 1296-1305, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38806708

RESUMEN

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.


Asunto(s)
Nociceptores , Dolor , Animales , Ratones , Dolor/inmunología , Dolor/metabolismo , Nociceptores/metabolismo , Transcriptoma , Ratones Endogámicos C57BL , Inflamación/inmunología , Masculino , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Animales de Enfermedad , Trombospondina 1/metabolismo , Trombospondina 1/genética , Piel/inmunología , Piel/metabolismo , Piel/patología , Zimosan , Análisis de la Célula Individual , Neuroinmunomodulación , Perfilación de la Expresión Génica , Neutrófilos/inmunología , Neutrófilos/metabolismo
8.
Cell ; 177(3): 711-721.e8, 2019 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-30982603

RESUMEN

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.


Asunto(s)
Proteínas Portadoras/metabolismo , Metionina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Peróxido de Hidrógeno/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Transición de Fase , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
Cell ; 177(3): 697-710.e17, 2019 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-30982600

RESUMEN

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.


Asunto(s)
Proteínas Portadoras/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Autofagia/efectos de los fármacos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Metionina/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología
10.
Cell ; 175(4): 962-972.e10, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30388453

RESUMEN

Many US immigrant populations develop metabolic diseases post immigration, but the causes are not well understood. Although the microbiome plays a role in metabolic disease, there have been no studies measuring the effects of US immigration on the gut microbiome. We collected stool, dietary recalls, and anthropometrics from 514 Hmong and Karen individuals living in Thailand and the United States, including first- and second-generation immigrants and 19 Karen individuals sampled before and after immigration, as well as from 36 US-born European American individuals. Using 16S and deep shotgun metagenomic DNA sequencing, we found that migration from a non-Western country to the United States is associated with immediate loss of gut microbiome diversity and function in which US-associated strains and functions displace native strains and functions. These effects increase with duration of US residence and are compounded by obesity and across generations.


Asunto(s)
Pueblo Asiatico , Emigración e Inmigración , Microbioma Gastrointestinal , Adulto , Bacteroides/aislamiento & purificación , Fibras de la Dieta/metabolismo , Emigrantes e Inmigrantes , Humanos , Metagenoma , Obesidad/epidemiología , Obesidad/microbiología , Prevotella/aislamiento & purificación , Estados Unidos
11.
Nat Immunol ; 21(12): 1496-1505, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33106668

RESUMEN

Transected axons typically fail to regenerate in the central nervous system (CNS), resulting in chronic neurological disability in individuals with traumatic brain or spinal cord injury, glaucoma and ischemia-reperfusion injury of the eye. Although neuroinflammation is often depicted as detrimental, there is growing evidence that alternatively activated, reparative leukocyte subsets and their products can be deployed to improve neurological outcomes. In the current study, we identify a unique granulocyte subset, with characteristics of an immature neutrophil, that had neuroprotective properties and drove CNS axon regeneration in vivo, in part via secretion of a cocktail of growth factors. This pro-regenerative neutrophil promoted repair in the optic nerve and spinal cord, demonstrating its relevance across CNS compartments and neuronal populations. Our findings could ultimately lead to the development of new immunotherapies that reverse CNS damage and restore lost neurological function across a spectrum of diseases.


Asunto(s)
Axones/metabolismo , Comunicación Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Regeneración Nerviosa , Neuronas/metabolismo , Neutrófilos/metabolismo , Animales , Biomarcadores , Plasticidad de la Célula/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Sistema Nervioso Central/inmunología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Ratones , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Nervio Óptico/inmunología , Nervio Óptico/metabolismo , Receptores de Interleucina-8B/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Transcriptoma , Zimosan/metabolismo , Zimosan/farmacología
12.
Cell ; 165(1): 100-110, 2016 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-26924577

RESUMEN

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.


Asunto(s)
Sinapsis Inmunológicas , Linfocitos T Citotóxicos/fisiología , Animales , Fenómenos Biomecánicos , Degranulación de la Célula , Línea Celular Tumoral , Ratones , Perforina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología
13.
Cell ; 164(3): 365-77, 2016 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-26806130

RESUMEN

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remains obscure. Here, we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, T cell receptor (TCR)αß, and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a, and CD103, these cells share a gene-expression signature distinct from those of conventional NK cells, T cells, and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15 deficiency, but not Nfil3 deficiency, results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type-1-like innate lymphoid cells and type 1 innate-like T cells.


Asunto(s)
Linfocitos/inmunología , Neoplasias Mamarias Experimentales/inmunología , Monitorización Inmunológica , Subgrupos de Linfocitos T/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Granzimas/metabolismo , Interleucina-15/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo
14.
Cell ; 162(2): 314-327, 2015 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-26144317

RESUMEN

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/ultraestructura , Virus de la Estomatitis Vesicular Indiana/química , Proteínas Virales/química , Proteínas Virales/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transcripción Genética
15.
Nature ; 629(8011): 323-328, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38720039

RESUMEN

The emergence of quasiparticles in quantum many-body systems underlies the rich phenomenology in many strongly interacting materials. In the context of doped Mott insulators, magnetic polarons are quasiparticles that usually arise from an interplay between the kinetic energy of doped charge carriers and superexchange spin interactions1-8. However, in kinetically frustrated lattices, itinerant spin polarons-bound states of a dopant and a spin flip-have been theoretically predicted even in the absence of superexchange coupling9-14. Despite their important role in the theory of kinetic magnetism, a microscopic observation of these polarons is lacking. Here we directly image itinerant spin polarons in a triangular-lattice Hubbard system realized with ultracold atoms, revealing enhanced antiferromagnetic correlations in the local environment of a hole dopant. In contrast, around a charge dopant, we find ferromagnetic correlations, a manifestation of the elusive Nagaoka effect15,16. We study the evolution of these correlations with interactions and doping, and use higher-order correlation functions to further elucidate the relative contributions of superexchange and kinetic mechanisms. The robustness of itinerant spin polarons at high temperature paves the way for exploring potential mechanisms for hole pairing and superconductivity in frustrated systems10,11. Furthermore, our work provides microscopic insights into related phenomena in triangular-lattice moiré materials17-20.

16.
Nature ; 626(7997): 119-127, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38200310

RESUMEN

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.


Asunto(s)
Núcleo Celular , Complejo I de Transporte de Electrón , Peces , Genes Letales , Especiación Genética , Hibridación Genética , Proteínas Mitocondriales , Animales , Alelos , Complejo I de Transporte de Electrón/genética , Peces/clasificación , Peces/embriología , Peces/genética , Peces/crecimiento & desarrollo , Homocigoto , Genes Letales/genética , Especificidad de la Especie , Desarrollo Embrionario/genética , Proteínas Mitocondriales/genética , Núcleo Celular/genética , Heterocigoto , Evolución Molecular
17.
Nature ; 630(8017): 752-761, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38867045

RESUMEN

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.


Asunto(s)
Disparidad de Par Base , Daño del ADN , ADN de Cadena Simple , Análisis de Secuencia de ADN , Imagen Individual de Molécula , Humanos , Envejecimiento/genética , Desaminasas APOBEC/genética , Desaminasas APOBEC/metabolismo , Disparidad de Par Base/genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citosina/metabolismo , Desaminación , Daño del ADN/genética , Reparación de la Incompatibilidad de ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Genoma Mitocondrial/genética , Mutación , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Imagen Individual de Molécula/métodos , Masculino , Femenino
18.
Nature ; 625(7993): 92-100, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38057664

RESUMEN

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders1-4, but attempts to assess constraint for non-protein-coding regions have proved more difficult. Here we aggregate, process and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD)-the largest public open-access human genome allele frequency reference dataset-and use it to build a genomic constraint map for the whole genome (genomic non-coding constraint of haploinsufficient variation (Gnocchi)). We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation. As expected, the average constraint for protein-coding sequences is stronger than that for non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants that are implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, which in turn suggests that non-coding constraint can aid the identification of constrained genes that are as yet unrecognized by current gene constraint metrics. We demonstrate that this genome-wide constraint map improves the identification and interpretation of functional human genetic variation.


Asunto(s)
Genoma Humano , Genómica , Modelos Genéticos , Mutación , Humanos , Acceso a la Información , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Frecuencia de los Genes , Genoma Humano/genética , Mutación/genética , Selección Genética
19.
Nature ; 629(8014): 1174-1181, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38720073

RESUMEN

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.


Asunto(s)
Fosfotirosina , Proteínas Tirosina Quinasas , Especificidad por Sustrato , Tirosina , Animales , Humanos , Secuencias de Aminoácidos , Evolución Molecular , Espectrometría de Masas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteómica , Transducción de Señal , Dominios Homologos src , Tirosina/metabolismo , Tirosina/química
20.
Mol Cell ; 82(1): 60-74.e5, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34995509

RESUMEN

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.


Asunto(s)
Gluconeogénesis , Glucosa/deficiencia , Histonas/metabolismo , Metabolismo de los Lípidos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Metabolismo de los Lípidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismo
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