RESUMEN
Lymphocytes spanning the entire innate-adaptive spectrum can stably reside in tissues and constitute an integral component of the local defense network against immunological challenges. In tight interactions with the epithelium and endothelium, tissue-resident lymphocytes sense antigens and alarmins elicited by infectious microbes and abiotic stresses at barrier sites and mount effector responses to restore tissue homeostasis. Of note, such a host cell-directed immune defense system has been recently demonstrated to surveil epithelial cell transformation and carcinoma development, as well as cancer cell metastasis at selected distant organs, and thus represents a primordial cancer immune defense module. Here we review how distinct lineages of tissue-resident innate lymphoid cells, innate-like T cells, and adaptive T cells participate in a form of multilayered cancer immunity in murine models and patients, and how their convergent effector programs may be targeted through both shared and private regulatory pathways for cancer immunotherapy.
Asunto(s)
Inmunidad Innata , Neoplasias , Humanos , Animales , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos/inmunología , Linfocitos/metabolismo , Microambiente Tumoral/inmunología , Inmunidad Adaptativa , Inmunoterapia/métodosRESUMEN
Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.
Asunto(s)
Células Progenitoras Endoteliales , Proteínas Supresoras de Tumor , Animales , Humanos , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa/genética , Macrófagos Asociados a Tumores/metabolismo , Células Progenitoras Endoteliales/metabolismo , Receptor de Proteína C Endotelial , Diana Mecanicista del Complejo 1 de la Rapamicina , Neovascularización Patológica , MamíferosRESUMEN
Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.
Asunto(s)
Linfocitos T CD8-positivos , Serotonina , Linfocitos T CD8-positivos/metabolismo , Serotonina/metabolismo , Serotonina/farmacología , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Transducción de Señal/fisiología , Células Th17/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno/metabolismo , Glucólisis/fisiología , L-Lactato Deshidrogenasa/deficiencia , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.
Asunto(s)
Adipocitos , Proteínas de Unión al Calcio , Metabolismo de los Lípidos , Proteínas de la Membrana , Animales , Femenino , Humanos , Ratones , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Placenta , Triglicéridos/metabolismo , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/metabolismo , Ácidos Grasos/metabolismo , Hipotermia/metabolismo , TermogénesisRESUMEN
T cell responses are guided by cytokines that induce transcriptional regulators, which ultimately control differentiation of effector and memory T cells. However, it is unknown how the activities of these molecular regulators are coordinated and integrated during the differentiation process. Using genetic approaches and transcriptional profiling of antigen-specific CD8(+) T cells, we reveal a common program of effector differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. The loss of both T-bet and Blimp-1 leads to abrogated cytotoxic function and ectopic IL-17 production in CD8(+) T cells. Overall, our data reveal two major overlapping pathways of effector differentiation governed by the availability of Blimp-1 and T-bet and suggest a model for cytokine-induced transcriptional changes that combine, quantitatively and qualitatively, to promote robust effector CD8(+) T cell differentiation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Interleucina-12/inmunología , Interleucina-2/inmunología , Proteínas de Dominio T Box/inmunología , Factores de Transcripción/inmunología , Animales , Infecciones por Arenaviridae/inmunología , Inmunoprecipitación de Cromatina , Citocinas/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Subtipo H1N1 del Virus de la Influenza A , Interleucina-17/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones , Infecciones por Orthomyxoviridae/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT4/inmunología , Factor de Transcripción STAT5/inmunología , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/fisiología , Factor de Transcripción AP-1/metabolismo , Virus Vaccinia/inmunología , Vaccinia/inmunología , Inmunidad Adaptativa , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Memoria Inmunológica/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Oncogénica p65(gag-jun) , Transducción de Señal/genética , Factor de Transcripción AP-1/genéticaRESUMEN
T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos T CD8-positivos/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Biomarcadores/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Femenino , Granzimas/genética , Granzimas/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/biosíntesis , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Coriomeningitis Linfocítica/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
Fermi liquid theory forms the basis for our understanding of the majority of metals: their resistivity arises from the scattering of well defined quasiparticles at a rate where, in the low-temperature limit, the inverse of the characteristic time scale is proportional to the square of the temperature. However, various quantum materials1-15-notably high-temperature superconductors1-10-exhibit strange-metallic behaviour with a linear scattering rate in temperature, deviating from this central paradigm. Here we show the unexpected signatures of strange metallicity in a bosonic system for which the quasiparticle concept does not apply. Our nanopatterned YBa2Cu3O7-δ (YBCO) film arrays reveal linear-in-temperature and linear-in-magnetic field resistance over extended temperature and magnetic field ranges. Notably, below the onset temperature at which Cooper pairs form, the low-field magnetoresistance oscillates with a period dictated by the superconducting flux quantum, h/2e (e, electron charge; h, Planck's constant). Simultaneously, the Hall coefficient drops and vanishes within the measurement resolution with decreasing temperature, indicating that Cooper pairs instead of single electrons dominate the transport process. Moreover, the characteristic time scale τ in this bosonic system follows a scale-invariant relation without an intrinsic energy scale: h/τ ≈ a(kBT + γµBB), where h is the reduced Planck's constant, a is of order unity7,8,11,12, kB is Boltzmann's constant, T is temperature, µB is the Bohr magneton and γ ≈ 2. By extending the reach of strange-metal phenomenology to a bosonic system, our results suggest that there is a fundamental principle governing their transport that transcends particle statistics.
Asunto(s)
Electrones , Superconductividad , Campos Magnéticos , Metales , TemperaturaRESUMEN
Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Inmunoterapia Adoptiva , Subunidad gamma Común de Receptores de Interleucina , Neoplasias , Receptores de Interleucina-9 , Proteínas Recombinantes de Fusión , Linfocitos T , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Inmunoterapia Adoptiva/métodos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Interleucinas/genética , Interleucinas/inmunología , Melanoma/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias Pancreáticas/inmunología , Receptores de Interleucina-9/genética , Receptores de Interleucina-9/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Transcripción STAT/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Receptor de Muerte Celular Programada 1 , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Quimioterapia Combinada , Humanos , Subunidad gamma Común de Receptores de Interleucina , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2 , Subunidad beta del Receptor de Interleucina-2 , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Factor 1 de Transcripción de Linfocitos TRESUMEN
The nucleosome remodeling and histone deacetylase (NuRD) complex physically associates with BCL11B to regulate murine T-cell development. However, the function of NuRD complex in mature T cells remains unclear. Here, we characterize the fate and metabolism of human T cells in which key subunits of the NuRD complex or BCL11B are ablated. BCL11B and the NuRD complex bind to each other and repress natural killer (NK)-cell fate in T cells. In addition, T cells upregulate the NK cell-associated receptors and transcription factors, lyse NK-cell targets, and are reprogrammed into NK-like cells (ITNKs) upon deletion of MTA2, MBD2, CHD4, or BCL11B. ITNKs increase OPA1 expression and exhibit characteristically elongated mitochondria with augmented oxidative phosphorylation (OXPHOS) activity. OPA1-mediated elevated OXPHOS enhances cellular acetyl-CoA levels, thereby promoting the reprogramming efficiency and antitumor effects of ITNKs via regulating H3K27 acetylation at specific targets. In conclusion, our findings demonstrate that the NuRD complex and BCL11B cooperatively maintain T-cell fate directly by repressing NK cell-associated transcription and indirectly through a metabolic-epigenetic axis, providing strategies to improve the reprogramming efficiency and antitumor effects of ITNKs.
Asunto(s)
Histonas , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Animales , Humanos , Ratones , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Dinámicas Mitocondriales , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Foxp3(+) regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.
Asunto(s)
Tejido Adiposo/citología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores Reguladores del Interferón/metabolismo , Interleucinas/metabolismo , Linfocitos T Reguladores/citología , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular/fisiología , Humanos , Interleucina-33 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/metabolismo , Obesidad/metabolismo , PPAR gamma/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Agonismo Parcial de Drogas , Interleucina-2/análogos & derivados , Interleucina-2/agonistas , Proteínas Mutantes/farmacología , Células Madre/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/inmunología , Interleucina-2/química , Interleucina-2/genética , Melanoma/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Factor de Transcripción STAT5/metabolismo , Células Madre/citología , Factor 1 de Transcripción de Linfocitos T/metabolismo , Investigación Biomédica TraslacionalRESUMEN
G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.
Asunto(s)
Receptores Acoplados a Proteínas G , Animales , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Ratones , Células HEK293 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Isoproterenol/farmacología , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Morfina/farmacología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Técnicas Biosensibles/métodosRESUMEN
In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.
Asunto(s)
Proteína BRCA1 , Proteínas de Ciclo Celular , Ratones Noqueados , Oocitos , Oocitos/metabolismo , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Femenino , Ratones , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Meiosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Roturas del ADN de Doble Cadena , Emparejamiento Cromosómico/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/genética , Recombinación Genética , Recombinación Homóloga , Inestabilidad GenómicaRESUMEN
Clonal hematopoiesis (CH) represents the clonal expansion of hematopoietic stem cells and their progeny driven by somatic mutations. Accurate risk assessment of CH is critical for disease prevention and clinical decision-making. The size of CH has been showed to associate with higher disease risk, yet, factors influencing the size of CH are unknown. In addition, the characteristics of CH in long-lived individuals are not well documented. Here, we report an in-depth analysis of CH in longevous (≥90 y old) and common (60~89 y old) elderly groups. Utilizing targeted deep sequencing, we found that the development of CH is closely related to age and the expression of aging biomarkers. The longevous elderly group exhibited a significantly higher incidence of CH and significantly higher frequency of TET2 and ASXL1 mutations, suggesting that certain CH could be beneficial to prolong life. Intriguingly, the size of CH neither correlates significantly to age, in the range of 60 to 110 y old, nor to the expression of aging biomarkers. Instead, we identified a strong correlation between large CH size and the number of mutations per individual. These findings provide a risk assessment biomarker for CH and also suggest that the evolution of the CH is influenced by factor(s) in addition to age.
Asunto(s)
Hematopoyesis Clonal , Hematopoyesis , Humanos , Anciano , Hematopoyesis Clonal/genética , Hematopoyesis/genética , Envejecimiento/genética , Mutación , BiomarcadoresRESUMEN
Natural or engineered peptides serve important biological functions. A general approach to achieve chemical-dependent activation of short peptides will be valuable for spatial and temporal control of cellular processes. Here we present a pair of chemically activated protein domains (CAPs) for controlling the accessibility of both the N- and C-terminal portion of a peptide. CAPs were developed through directed evolution of an FK506-binding protein. By fusing a peptide to one or both CAPs, the function of the peptide is blocked until a small molecule displaces them from the FK506-binding protein ligand-binding site. We demonstrate that CAPs are generally applicable to a range of short peptides, including a protease cleavage site, a dimerization-inducing heptapeptide, a nuclear localization signal peptide, and an opioid peptide, with a chemical dependence up to 156-fold. We show that the CAPs system can be utilized in cell cultures and multiple organs in living animals.
Asunto(s)
Péptido Hidrolasas , Péptidos , Animales , Péptidos/química , Endopeptidasas/metabolismo , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
Perennial trees must maintain stem growth throughout their entire lifespan to progressively increase in size as they age. The overarching question of the molecular mechanisms that govern stem perennial growth in trees remains largely unanswered. Here we deciphered the genetic architecture that underlies perennial growth trajectories using genome-wide association studies (GWAS) for measures of growth traits across years in a natural population of Populus tomentosa. By analyzing the stem growth trajectory, we identified PtoP4H9, encoding prolyl 4-hydroxylase 9, which is responsible for the natural variation in the growth rate of diameter at breast height (DBH) across years. Quantifying the dynamic genetic contribution of PtoP4H9 loci to stem growth showed that PtoP4H9 played a pivotal role in stem growth regulation. Spatiotemporal expression analysis showed that PtoP4H9 was highly expressed in cambium tissues of poplars of various ages. Overexpression and knockdown of PtoP4H9 revealed that it altered cell expansion to regulate cell wall modification and mechanical characteristics, thereby promoting stem growth in Populus. We showed that natural variation in PtoP4H9 occurred in a BASIC PENTACYSTEINE transcription factor PtoBPC1-binding promoter element controlling PtoP4H9 expression. The geographic distribution of PtoP4H9 allelic variation was consistent with the modes of selection among populations. Altogether, our study provides important genetic insights into dynamic stem growth in Populus, and we confirmed PtoP4H9 as a potential useful marker for breeding or genetic engineering of poplars.
Asunto(s)
Populus , Estudio de Asociación del Genoma Completo , Prolil Hidroxilasas/genética , Prolil Hidroxilasas/metabolismo , Genes de Plantas , FenotipoRESUMEN
Collateral circulation is essential for blood resupply to the ischemic heart, which is dictated by the contractile phenotypic restoration of vascular smooth muscle cells (VSMC). Here we investigate whether S-nitrosylation of AMP-activated protein kinase (AMPK), a key regulator of the VSMC phenotype, impairs collateral circulation. In rats with collateral growth and development, nitroglycerin decreases coronary collateral blood flow (CCBF), inhibits vascular contractile phenotypic restoration, and increases myocardial infarct size, accompanied by reduced AMPK activity in the collateral zone. Nitric oxide (NO) S-nitrosylates human recombinant AMPKγ1 at cysteine 131 and decreases AMP sensitivity of AMPK. In VSMCs, exogenous expression of S-nitrosylation-resistant AMPKγ1 or deficient NO synthase (iNOS) prevents the disruption of VSMC reprogramming. Finally, hyperhomocysteinemia or hyperglycemia increases AMPKγ1 S-nitrosylation, prevents vascular contractile phenotypic restoration, reduces CCBF, and increases the infarct size of the heart in Apoe-/- mice, all of which is rescued in Apoe-/-/iNOSsm-/- mice or Apoe-/- mice with enforced expression of the AMPKγ1-C130A mutant following RI/MI. We conclude that nitrosative stress disrupts coronary collateral circulation during hyperhomocysteinemia or hyperglycemia through AMPK S-nitrosylation.