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1.
Nat Immunol ; 23(5): 757-767, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35437325

RESUMEN

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4+ and CD8+ T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies.


Asunto(s)
Linfocitos T CD8-positivos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Antígenos CD/inmunología , Complejo CD3/inmunología , Antígenos CD8/metabolismo , Antígenos de Histocompatibilidad Clase II , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteína del Gen 3 de Activación de Linfocitos
2.
Immunity ; 56(8): 1862-1875.e9, 2023 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-37478853

RESUMEN

Loss of oral tolerance (LOT) to gluten, driven by dendritic cell (DC) priming of gluten-specific T helper 1 (Th1) cell immune responses, is a hallmark of celiac disease (CeD) and can be triggered by enteric viral infections. Whether certain commensals can moderate virus-mediated LOT remains elusive. Here, using a mouse model of virus-mediated LOT, we discovered that the gut-colonizing protist Tritrichomonas (T.) arnold promotes oral tolerance and protects against reovirus- and murine norovirus-mediated LOT, independent of the microbiota. Protection was not attributable to antiviral host responses or T. arnold-mediated innate type 2 immunity. Mechanistically, T. arnold directly restrained the proinflammatory program in dietary antigen-presenting DCs, subsequently limiting Th1 and promoting regulatory T cell responses. Finally, analysis of fecal microbiomes showed that T. arnold-related Parabasalid strains are underrepresented in human CeD patients. Altogether, these findings will motivate further exploration of oral-tolerance-promoting protists in CeD and other immune-mediated food sensitivities.


Asunto(s)
Antígenos , Inmunidad Innata , Animales , Ratones , Humanos , Dieta , Glútenes , Células Dendríticas , Tolerancia Inmunológica
3.
Cell ; 171(3): 628-641.e26, 2017 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-29053969

RESUMEN

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.


Asunto(s)
Lesión Renal Aguda/patología , Asma/patología , Lesiones Traumáticas del Encéfalo/patología , Muerte Celular , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Lesión Renal Aguda/metabolismo , Animales , Apoptosis , Asma/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Isoenzimas/metabolismo , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Ratones , Modelos Moleculares , Oxazolidinonas/farmacología , Oxidación-Reducción , Proteínas de Unión a Fosfatidiletanolamina/química
4.
Nature ; 626(7998): 367-376, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38092041

RESUMEN

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.


Asunto(s)
Desarrollo Embrionario , Estratos Germinativos , Hematopoyesis , Saco Vitelino , Humanos , Implantación del Embrión , Endodermo/citología , Endodermo/embriología , Estratos Germinativos/citología , Estratos Germinativos/embriología , Saco Vitelino/citología , Saco Vitelino/embriología , Mesodermo/citología , Mesodermo/embriología , Células Madre Pluripotentes Inducidas/citología , Amnios/citología , Amnios/embriología , Cuerpos Embrioides/citología , Linaje de la Célula , Biología Evolutiva/métodos , Biología Evolutiva/tendencias
5.
Mol Cell ; 82(21): 4001-4017.e7, 2022 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-36265488

RESUMEN

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.


Asunto(s)
ARN Largo no Codificante , Homeostasis del Telómero , Cromatina/genética , Proteómica , Telómero/genética , Telómero/metabolismo , ARN Largo no Codificante/genética , Homeostasis
6.
Immunity ; 49(4): 740-753.e7, 2018 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-30314759

RESUMEN

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.


Asunto(s)
Caspasas/inmunología , Endotoxinas/inmunología , Proteína HMGB1/inmunología , Piroptosis/inmunología , Sepsis/inmunología , Animales , Caspasas/genética , Caspasas/metabolismo , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Endotoxinas/metabolismo , Células HEK293 , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada/inmunología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Sepsis/genética , Sepsis/metabolismo , Células THP-1
7.
Mol Cell ; 75(1): 117-130.e6, 2019 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-31101499

RESUMEN

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.


Asunto(s)
Aberraciones Cromosómicas/efectos de la radiación , ADN Glicosilasas/genética , Reparación del ADN/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Guanina/análogos & derivados , Telómero/efectos de la radiación , División Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Daño del ADN , ADN Glicosilasas/deficiencia , Replicación del ADN/efectos de la radiación , Expresión Génica , Guanina/agonistas , Guanina/biosíntesis , Células HeLa , Humanos , Luz/efectos adversos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Optogenética , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoblastos/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Oxígeno Singlete/agonistas , Oxígeno Singlete/metabolismo , Telómero/metabolismo , Homeostasis del Telómero/efectos de la radiación
8.
Mol Cell ; 76(1): 11-26.e7, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31400850

RESUMEN

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Homeostasis del Telómero , Telómero/metabolismo , Autofagia , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proliferación Celular , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Recombinación Homóloga , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligasas/genética , Ligasas/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Estabilidad Proteica , Proteínas de Unión al ARN/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Transducción de Señal , Sumoilación , Telómero/genética , Telómero/patología
9.
N Engl J Med ; 388(8): 719-732, 2023 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-36795891

RESUMEN

BACKGROUND: In a phase 2 study, rucaparib, an inhibitor of poly(ADP-ribose) polymerase (PARP), showed a high level of activity in patients who had metastatic, castration-resistant prostate cancer associated with a deleterious BRCA alteration. Data are needed to confirm and expand on the findings of the phase 2 study. METHODS: In this randomized, controlled, phase 3 trial, we enrolled patients who had metastatic, castration-resistant prostate cancer with a BRCA1, BRCA2, or ATM alteration and who had disease progression after treatment with a second-generation androgen-receptor pathway inhibitor (ARPI). We randomly assigned the patients in a 2:1 ratio to receive oral rucaparib (600 mg twice daily) or a physician's choice control (docetaxel or a second-generation ARPI [abiraterone acetate or enzalutamide]). The primary outcome was the median duration of imaging-based progression-free survival according to independent review. RESULTS: Of the 4855 patients who had undergone prescreening or screening, 270 were assigned to receive rucaparib and 135 to receive a control medication (intention-to-treat population); in the two groups, 201 patients and 101 patients, respectively, had a BRCA alteration. At 62 months, the duration of imaging-based progression-free survival was significantly longer in the rucaparib group than in the control group, both in the BRCA subgroup (median, 11.2 months and 6.4 months, respectively; hazard ratio, 0.50; 95% confidence interval [CI], 0.36 to 0.69) and in the intention-to-treat group (median, 10.2 months and 6.4 months, respectively; hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001 for both comparisons). In an exploratory analysis in the ATM subgroup, the median duration of imaging-based progression-free survival was 8.1 months in the rucaparib group and 6.8 months in the control group (hazard ratio, 0.95; 95% CI, 0.59 to 1.52). The most frequent adverse events with rucaparib were fatigue and nausea. CONCLUSIONS: The duration of imaging-based progression-free survival was significantly longer with rucaparib than with a control medication among patients who had metastatic, castration-resistant prostate cancer with a BRCA alteration. (Funded by Clovis Oncology; TRITON3 ClinicalTrials.gov number, NCT02975934.).


Asunto(s)
Antineoplásicos , Neoplasias de la Próstata Resistentes a la Castración , Humanos , Masculino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Indoles/uso terapéutico , Supervivencia sin Progresión , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/secundario , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Antagonistas de Andrógenos/uso terapéutico , Antineoplásicos/uso terapéutico , Docetaxel/uso terapéutico , Progresión de la Enfermedad , Genes BRCA1 , Genes BRCA2
10.
EMBO Rep ; 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39191946

RESUMEN

Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1.

11.
PLoS Genet ; 19(4): e1010710, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37068109

RESUMEN

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.


Asunto(s)
Síndrome de Prader-Willi , Ratones , Animales , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Secreción de Insulina/genética , Chaperón BiP del Retículo Endoplásmico , Regulación hacia Abajo , Proteómica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Insulina/genética , Insulina/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo
12.
Proc Natl Acad Sci U S A ; 120(25): e2218896120, 2023 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-37327313

RESUMEN

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.


Asunto(s)
Ferroptosis , Proteínas de Unión a Fosfatidiletanolamina , Glutatión/metabolismo , Hierro/metabolismo , Peroxidación de Lípido , Lípidos , Oxidación-Reducción , Proteínas de Unión a Fosfatidiletanolamina/antagonistas & inhibidores
13.
Immunity ; 45(2): 374-88, 2016 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-27496732

RESUMEN

Although tumor-specific T cells recognize cancer cells, they are often rendered dysfunctional due to an immunosuppressive microenvironment. Here we showed that T cells demonstrated persistent loss of mitochondrial function and mass when infiltrating murine and human tumors, an effect specific to the tumor microenvironment and not merely caused by activation. Tumor-infiltrating T cells showed a progressive loss of PPAR-gamma coactivator 1α (PGC1α), which programs mitochondrial biogenesis, induced by chronic Akt signaling in tumor-specific T cells. Reprogramming tumor-specific T cells through enforced expression of PGC1α resulted in superior intratumoral metabolic and effector function. Our data support a model in which signals in the tumor microenvironment repress T cell oxidative metabolism, resulting in effector cells with metabolic needs that cannot be met. Our studies also suggest that modulation or reprogramming of the altered metabolism of tumor-infiltrating T cells might represent a potential strategy to reinvigorate dysfunctional T cells for cancer treatment.


Asunto(s)
Neoplasias del Colon/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Reprogramación Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales , Proteína Oncogénica v-akt/metabolismo , Estrés Oxidativo , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Transducción de Señal , Microambiente Tumoral
14.
Nucleic Acids Res ; 51(10): 4881-4898, 2023 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-36971122

RESUMEN

UV-damaged DNA-binding protein (UV-DDB) is a heterodimeric protein, consisting of DDB1 and DDB2 subunits, that works to recognize DNA lesions induced by UV damage during global genome nucleotide excision repair (GG-NER). Our laboratory previously discovered a non-canonical role for UV-DDB in the processing of 8-oxoG, by stimulating 8-oxoG glycosylase, OGG1, activity 3-fold, MUTYH activity 4-5-fold, and APE1 (apurinic/apyrimidinic endonuclease 1) activity 8-fold. 5-hydroxymethyl-deoxyuridine (5-hmdU) is an important oxidation product of thymidine which is removed by single-strand selective monofunctional DNA glycosylase (SMUG1). Biochemical experiments with purified proteins indicated that UV-DDB stimulates the excision activity of SMUG1 on several substrates by 4-5-fold. Electrophoretic mobility shift assays indicated that UV-DDB displaced SMUG1 from abasic site products. Single-molecule analysis revealed that UV-DDB decreases the half-life of SMUG1 on DNA by ∼8-fold. Immunofluorescence experiments demonstrated that cellular treatment with 5-hmdU (5 µM for 15 min), which is incorporated into DNA during replication, produces discrete foci of DDB2-mCherry, which co-localize with SMUG1-GFP. Proximity ligation assays supported a transient interaction between SMUG1 and DDB2 in cells. Poly(ADP)-ribose accumulated after 5-hmdU treatment, which was abrogated with SMUG1 and DDB2 knockdown. These data support a novel role for UV-DDB in the processing of the oxidized base, 5-hmdU.


Asunto(s)
Daño del ADN , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Reparación del ADN , ADN/química , Timidina , Rayos Ultravioleta
15.
Kidney Int ; 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38901605

RESUMEN

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

16.
PLoS Pathog ; 18(3): e1010395, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35271686

RESUMEN

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1ß and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae , Animales , Interferones/inmunología , Pulmón , Macrófagos Alveolares/inmunología , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Primates , Piroptosis
17.
Am J Pathol ; 193(2): 201-212, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36414085

RESUMEN

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.


Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , ADN Mitocondrial , Neuronas/metabolismo , Dendritas/metabolismo , Proteínas Quinasas/genética , ADN Polimerasa gamma/genética
18.
J Exp Biol ; 227(15)2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39111742

RESUMEN

Wind-hovering birds exhibit remarkable steadiness in flight, achieved through the morphing of their wings and tail. We analysed the kinematics of two nankeen kestrels (Falco cenchroides) engaged in steady wind-hovering flights in a smooth flow wind tunnel. Motion-tracking cameras were used to capture the movements of the birds as they maintained their position. The motion of the birds' head and body, and the morphing motions of their wings and tail were tracked and analysed using correlation methods. The results revealed that wing sweep, representing the flexion/extension movement of the wing, played a significant role in wing motion. Additionally, correlations between different independent degrees of freedom (DoF), including wing and tail coupling, were observed. These kinematic couplings indicate balancing of forces and moments necessary for steady wind hovering. Variation in flight behaviour between the two birds highlighted the redundancy of DoF and the versatility of wing morphing in achieving control. This study provides insights into fixed-wing craft flight control from the avian world and may inspire novel flight control strategies for future fixed-wing aircraft.


Asunto(s)
Falconiformes , Vuelo Animal , Cola (estructura animal) , Alas de Animales , Animales , Vuelo Animal/fisiología , Alas de Animales/fisiología , Alas de Animales/anatomía & histología , Fenómenos Biomecánicos , Cola (estructura animal)/fisiología , Cola (estructura animal)/anatomía & histología , Falconiformes/fisiología , Falconiformes/anatomía & histología , Viento
19.
Arterioscler Thromb Vasc Biol ; 43(10): 2023-2029, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37675635

RESUMEN

BACKGROUND: We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. METHODS: Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. RESULTS: Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. CONCLUSIONS: This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.


Asunto(s)
Ferroptosis , Enfermedades Musculares , Enfermedad Arterial Periférica , Células Satélite del Músculo Esquelético , Humanos , Ferroptosis/genética , Células Satélite del Músculo Esquelético/metabolismo , Transcriptoma , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Peroxidación de Lípido/fisiología , Hierro/metabolismo , Enfermedad Arterial Periférica/genética , Isquemia
20.
Mol Cell ; 64(2): 376-387, 2016 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-27720644

RESUMEN

Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the ß-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of ß-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.


Asunto(s)
Reparación del ADN , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Daño del ADN , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Puntos Cuánticos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Imagen Individual de Molécula , Rayos Ultravioleta
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