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1.
Cell ; 185(16): 3008-3024.e16, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35870449

RESUMEN

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.


Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Animales , Sistemas CRISPR-Cas/genética , Edición Génica , Genoma , Ratones , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Transgenes
2.
Nat Immunol ; 23(11): 1577-1587, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36271146

RESUMEN

Aberrant RNA splicing in keratinocytes drives inflammatory skin disorders. In the present study, we found that the RNA helicase DDX5 was downregulated in keratinocytes from the inflammatory skin lesions in patients with atopic dermatitis and psoriasis, and that mice with keratinocyte-specific deletion of Ddx5 (Ddx5∆KC) were more susceptible to cutaneous inflammation. Inhibition of DDX5 expression in keratinocytes was induced by the cytokine interleukin (IL)-17D through activation of the CD93-p38 MAPK-AKT-SMAD2/3 signaling pathway and led to pre-messenger RNA splicing events that favored the production of membrane-bound, intact IL-36 receptor (IL-36R) at the expense of soluble IL-36R (sIL-36R) and to the selective amplification of IL-36R-mediated inflammatory responses and cutaneous inflammation. Restoration of sIL-36R in Ddx5∆KC mice with experimental atopic dermatitis or psoriasis suppressed skin inflammation and alleviated the disease phenotypes. These findings indicate that IL-17D modulation of DDX5 expression controls inflammation in keratinocytes during inflammatory skin diseases.


Asunto(s)
Dermatitis Atópica , Interleucina-27 , Psoriasis , Ratones , Animales , Interleucina-27/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Queratinocitos/metabolismo , Piel/patología , Psoriasis/genética , Psoriasis/patología , Inflamación/metabolismo
3.
Immunity ; 56(5): 897-900, 2023 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-37163988

RESUMEN

How pattern recognition receptors NOD1 and NOD2 sense bacterial muropeptides from extracellular bacteria to drive keratinocyte inflammation remains unclear. In this issue of Immunity, Bharadwaj et al. show that the solute carrier 46A2 (SLC46A2) delivers DAP-muropeptides into the cytosol to drive NOD1 activation in keratinocytes and elicit skin inflammation during psoriasis.


Asunto(s)
Inflamación , Receptores de Reconocimiento de Patrones , Humanos , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo
4.
Immunity ; 56(11): 2555-2569.e5, 2023 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-37967531

RESUMEN

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 , Mamíferos
5.
Immunity ; 55(11): 2044-2058.e5, 2022 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-36288724

RESUMEN

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.


Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Animales , Ratones , Macrófagos Asociados a Tumores , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Linfocitos T Citotóxicos , Células Dendríticas
6.
Nature ; 619(7970): 616-623, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37380769

RESUMEN

In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3-6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent 'winner' cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39-14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.


Asunto(s)
Competencia Celular , Técnicas de Reprogramación Celular , Inmunidad Innata , Neoplasias , Macrófagos Asociados a Tumores , Animales , Ratones , Aminoácidos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Competencia Celular/genética , Competencia Celular/inmunología , Proteínas en la Dieta/farmacología , Modelos Animales de Enfermedad , GTP Fosfohidrolasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo
7.
Nat Immunol ; 16(10): 1044-50, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26280998

RESUMEN

The cellular and molecular events that drive the early development of innate lymphoid cells (ILCs) remain poorly understood. We show that the transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILPs) expressing high amounts of TCF-1. EILPs lacked efficient T and B lymphocyte potential but efficiently gave rise to NK cells and all known adult helper ILC lineages, indicating that they are the earliest ILC-committed progenitors identified so far. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILPs provides a basis for deciphering additional signals that specify ILC fate.


Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Inmunidad Innata , Linfocitos/citología , Linfocitos/inmunología , Factor 1 de Transcripción de Linfocitos T/genética , Regulación hacia Arriba , Animales , Células Cultivadas , Citometría de Flujo , Ratones , Análisis por Micromatrices , Factor 1 de Transcripción de Linfocitos T/metabolismo
8.
Nature ; 598(7879): 129-136, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34616068

RESUMEN

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.


Asunto(s)
Cerebro/citología , Cerebro/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Atlas como Asunto , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades del Sistema Nervioso/genética , Neuroglía/clasificación , Neuroglía/metabolismo , Neuronas/clasificación , Neuronas/metabolismo , Análisis de Secuencia de ADN , Análisis de la Célula Individual
9.
PLoS Pathog ; 20(5): e1012228, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38739679

RESUMEN

The arthropod exoskeleton provides protection and support and is vital for survival and adaption. The integrity and mechanical properties of the exoskeleton are often impaired after pathogenic infection; however, the detailed mechanism by which infection affects the exoskeleton remains largely unknown. Here, we report that the damage to the shrimp exoskeleton is caused by modulation of host lipid profiles after infection with white spot syndrome virus (WSSV). WSSV infection disrupts the mechanical performance of the exoskeleton by inducing the expression of a chitinase (Chi2) in the sub-cuticle epidermis and decreasing the cuticle chitin content. The induction of Chi2 expression is mediated by a nuclear receptor that can be activated by certain enriched long-chain saturated fatty acids after infection. The damage to the exoskeleton, an aftereffect of the induction of host lipogenesis by WSSV, significantly impairs the motor ability of shrimp. Blocking the WSSV-caused lipogenesis restored the mechanical performance of the cuticle and improved the motor ability of infected shrimp. Therefore, this study reveals a mechanism by which WSSV infection modulates shrimp internal metabolism resulting in phenotypic impairment, and provides new insights into the interactions between the arthropod host and virus.


Asunto(s)
Exoesqueleto , Metabolismo de los Lípidos , Penaeidae , Virus del Síndrome de la Mancha Blanca 1 , Animales , Penaeidae/virología , Penaeidae/metabolismo , Exoesqueleto/metabolismo , Exoesqueleto/virología , Virus del Síndrome de la Mancha Blanca 1/fisiología , Metabolismo de los Lípidos/fisiología , Interacciones Huésped-Patógeno , Lipogénesis/fisiología
10.
Nat Immunol ; 15(7): 667-75, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24859450

RESUMEN

CD4(+) follicular helper T cells (T(FH) cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4(+) T cells deficient in the transcription factor Foxp1 'preferentially' differentiated into T(FH) cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of T(FH) cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4(+) T cells partially resistant to blockade of the ICOS ligand (ICOSL) during T(FH) cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of T(FH) cell differentiation.


Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead/fisiología , Proteínas Represoras/fisiología , Linfocitos T Colaboradores-Inductores/citología , Animales , Linfocitos T CD4-Positivos/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Interleucinas/genética , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/fisiología
11.
Proc Natl Acad Sci U S A ; 120(29): e2215744120, 2023 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-37428911

RESUMEN

Hepatocellular carcinoma (HCC) takes the predominant malignancy of hepatocytes with bleak outcomes owing to high heterogeneity among patients. Personalized treatments based on molecular profiles will better improve patients' prognosis. Lysozyme (LYZ), a secretory protein with antibacterial function generally expressed in monocytes/macrophages, has been observed for the prognostic implications in different types of tumors. However, studies about the explicit applicative scenarios and mechanisms for tumor progression are still quite limited, especially for HCC. Here, based on the proteomic molecular classification data of early-stage HCC, we revealed that the LYZ level was elevated significantly in the most malignant HCC subtype and could serve as an independent prognostic predictor for HCC patients. Molecular profiles of LYZ-high HCCs were typical of those for the most malignant HCC subtype, with impaired metabolism, along with promoted proliferation and metastasis characteristics. Further studies demonstrated that LYZ tended to be aberrantly expressed in poorly differentiated HCC cells, which was regulated by STAT3 activation. LYZ promoted HCC proliferation and migration in both autocrine and paracrine manners independent of the muramidase activity through the activation of downstream protumoral signaling pathways via cell surface GRP78. Subcutaneous and orthotopic xenograft tumor models indicated that targeting LYZ inhibited HCC growth markedly in NOD/SCID mice. These results propose LYZ as a prognostic biomarker and therapeutic target for the subclass of HCC with an aggressive phenotype.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratones , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Muramidasa/metabolismo , Proteómica , Línea Celular Tumoral , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Procesos Neoplásicos , Biomarcadores de Tumor/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica
12.
Eur J Immunol ; 54(5): e2250279, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38356200

RESUMEN

Although biologics have achieved tremendous success in the treatment of psoriasis and revolutionized the clinical management of the disease, certain issues arise during treatments, including the phenotypic switch from psoriasis to other skin disorders and the recurrence of psoriasis after the cessation of biologic treatment. Here we provide a concise overview of the roles of keratinocytes in the pathogenesis of psoriasis, elucidate the involvement of keratinocytes in the phenotypic switch and relapse of psoriasis, and address the challenges encountered in both basic and clinical research on psoriasis.


Asunto(s)
Queratinocitos , Fenotipo , Psoriasis , Recurrencia , Psoriasis/inmunología , Psoriasis/patología , Humanos , Queratinocitos/inmunología , Animales
13.
Nat Immunol ; 14(12): 1277-84, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24185616

RESUMEN

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Proteína alfa Potenciadora de Unión a CCAAT/inmunología , Proteínas de Homeodominio/inmunología , Receptor Notch1/inmunología , Linfocitos T/inmunología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Expresión Génica/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Linfopoyesis/genética , Linfopoyesis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Unión Proteica/inmunología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Madre/inmunología , Células Madre/metabolismo , Linfocitos T/metabolismo , Factor de Transcripción HES-1
14.
Exp Cell Res ; 436(2): 113976, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38401687

RESUMEN

Glioma is the most common brain malignancy, characterized by high morbidity, high mortality, and treatment-resistance. Inverted CCAAT box Binding Protein of 90 kDa (ICBP90) has been reported to be involved in tumor progression and the maintenance of DNA methylation. Herein, we constructed ICBP90 over-expression and knockdown glioma cell lines, and found that ICBP90 knockdown inhibited glioma cell proliferation, migration, and invasion. ICBP90 silencing potentially enhanced cellular sensitivity to cis-platinum (DDP) and exacerbated DDP-induced pyroptosis, manifested by the elevated levels of gasdermin D-N-terminal and cleaved caspase 1; whereas, ICBP90 over-expression exhibited the opposite effects. Consistently, ICBP90 knockdown inhibited tumor growth in an in vivo mouse xenograft study using U251 cells stably expressing sh-ICBP90 and oe-ICBP90. Further experiments found that ICBP90 reduced the expression of Dickkopf 3 homolog (DKK3), a negative regulator of ß-catenin, by binding its promoter and inducing DNA methylation. ICBP90 knockdown prevented the nuclear translocation of ß-catenin and suppressed the expression of c-Myc and cyclin D1. Besides, DKK3 over-expression restored the effects of ICBP90 over-expression on cell proliferation, migration, invasion, and DDP sensitivity. Our findings suggest that ICBP90 inhibits the expression of DKK3 in glioma by maintaining DKK3 promoter methylation, thereby conducing to ICBP90-mediated carcinogenesis and drug insensitivity.


Asunto(s)
Glioma , beta Catenina , Humanos , Animales , Ratones , beta Catenina/metabolismo , Cisplatino/farmacología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metilación de ADN/genética , Epigénesis Genética/genética , Glioma/genética , Línea Celular Tumoral , Proliferación Celular/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo
15.
Nucleic Acids Res ; 51(D1): D460-D465, 2023 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-36124686

RESUMEN

Phase separation (PS) proteins form droplets to regulate myriad membraneless organelles (MLOs) and cellular pathways such as transcription, signaling transduction and protein degeneration. PS droplets are usually liquid-like and can convert to hydrogel/solid-like under certain conditions. The PS behavior of proteins is regulated by co-PS partners and mutations, modifications, oligomerizations, repeat regions and alternative splicing of the proteins. With growing interest in PS condensates and associated proteins, we established PhaSepDB 1.0, which provided experimentally verified PS proteins and MLO-related proteins. The past few years witnessed a surge in PS-related research works; thus, we kept updating PhaSepDB. The current PhaSepDB contains 1419 PS entries, 770 low-throughput MLO-related entries and 7303 high-throughput MLO-related entries. We provided more detailed annotations of PS proteins, including PS verification experiments, regions used in experiments, phase diagrams of different experimental conditions, droplet states, co-PS partners and PS regulatory information. We believe that researchers can go further in studying PS proteins with the updated PhaSepDB (http://db.phasep.pro/).


Asunto(s)
Fenómenos Biomecánicos , Células , Orgánulos , Proteínas , Orgánulos/química , Proteínas/química , Células/química , Células/citología
16.
EMBO J ; 39(22): e104748, 2020 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-33058207

RESUMEN

Programmable A > I RNA editing is a valuable tool for basic research and medicine. A variety of editors have been created, but a genetically encoded editor that is both precise and efficient has not been described to date. The trade-off between precision and efficiency is exemplified in the state of the art editor REPAIR, which comprises the ADAR2 deaminase domain fused to dCas13b. REPAIR is highly efficient, but also causes significant off-target effects. Mutations that weaken the deaminase domain can minimize the undesirable effects, but this comes at the expense of on-target editing efficiency. We have now overcome this dilemma by using a multipronged approach: We have chosen an alternative Cas protein (CasRx), inserted the deaminase domain into the middle of CasRx, and redirected the editor to the nucleus. The new editor created, dubbed REPAIRx, is precise yet highly efficient, outperforming various previous versions on both mRNA and nuclear RNA targets. Thus, REPAIRx markedly expands the RNA editing toolkit and illustrates a novel strategy for base editor optimization.


Asunto(s)
Edición Génica/métodos , Edición de ARN , ARN/metabolismo , Adenosina Desaminasa/genética , Células HEK293 , Humanos , Mutación , Proteínas de Unión al ARN/genética , Transcriptoma
17.
EMBO J ; 39(22): e104741, 2020 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-33058229

RESUMEN

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.


Asunto(s)
Citidina Desaminasa/genética , Proteínas/genética , Edición de ARN , Núcleo Celular/metabolismo , Células HEK293 , Humanos , ARN/química , ARN/metabolismo , ARN Guía de Kinetoplastida , Transcriptoma
18.
Am J Hum Genet ; 108(4): 583-596, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33798444

RESUMEN

The contribution of genome structural variation (SV) to quantitative traits associated with cardiometabolic diseases remains largely unknown. Here, we present the results of a study examining genetic association between SVs and cardiometabolic traits in the Finnish population. We used sensitive methods to identify and genotype 129,166 high-confidence SVs from deep whole-genome sequencing (WGS) data of 4,848 individuals. We tested the 64,572 common and low-frequency SVs for association with 116 quantitative traits and tested candidate associations using exome sequencing and array genotype data from an additional 15,205 individuals. We discovered 31 genome-wide significant associations at 15 loci, including 2 loci at which SVs have strong phenotypic effects: (1) a deletion of the ALB promoter that is greatly enriched in the Finnish population and causes decreased serum albumin level in carriers (p = 1.47 × 10-54) and is also associated with increased levels of total cholesterol (p = 1.22 × 10-28) and 14 additional cholesterol-related traits, and (2) a multi-allelic copy number variant (CNV) at PDPR that is strongly associated with pyruvate (p = 4.81 × 10-21) and alanine (p = 6.14 × 10-12) levels and resides within a structurally complex genomic region that has accumulated many rearrangements over evolutionary time. We also confirmed six previously reported associations, including five led by stronger signals in single nucleotide variants (SNVs) and one linking recurrent HP gene deletion and cholesterol levels (p = 6.24 × 10-10), which was also found to be strongly associated with increased glycoprotein level (p = 3.53 × 10-35). Our study confirms that integrating SVs in trait-mapping studies will expand our knowledge of genetic factors underlying disease risk.


Asunto(s)
Enfermedades Cardiovasculares/genética , Variación Estructural del Genoma/genética , Alelos , Colesterol/sangre , Variaciones en el Número de Copia de ADN/genética , Femenino , Finlandia , Genoma Humano/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Proteínas Mitocondriales/genética , Regiones Promotoras Genéticas/genética , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/genética , Ácido Pirúvico/metabolismo , Albúmina Sérica Humana/genética
19.
Gastroenterology ; 165(2): 402-413.e13, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-36894035

RESUMEN

BACKGROUND & AIMS: Diagnosing gastric cancer (GC) while the disease remains eligible for surgical resection is challenging. In view of this clinical challenge, novel and robust biomarkers for early detection thus improving prognosis of GC are necessary. The present study is to develop a blood-based long noncoding RNA (LR) signature for the early-detection of GC. METHODS: The present 3-step study incorporated data from 2141 patients, including 888 with GC, 158 with chronic atrophic gastritis, 193 with intestinal metaplasia, 501 healthy donors, and 401 with other gastrointestinal cancers. The LR profile of stage I GC tissue samples were analyzed using transcriptomic profiling in discovery phase. The extracellular vesicle (EV)-derived LR signature was identified with a training cohort (n = 554) and validated with 2 external cohorts (n = 429 and n = 504) and a supplemental cohort (n = 69). RESULTS: In discovery phase, one LR (GClnc1) was found to be up-regulated in both tissue and circulating EV samples with an area under the curve (AUC) of 0.9369 (95% confidence interval [CI], 0.9073-0.9664) for early-stage GC (stage I/II). The diagnostic performance of this biomarker was further confirmed in 2 external validation cohorts (Xi'an cohort, AUC: 0.8839; 95% CI: 0.8336-0.9342; Beijing cohort, AUC: 0.9018; 95% CI: 0.8597-0.9439). Moreover, EV-derived GClnc1 robustly distinguished early-stage GC from precancerous lesions (chronic atrophic gastritis and intestinal metaplasia) and GC with negative traditional gastrointestinal biomarkers (CEA, CA72-4, and CA19-9). The low levels of this biomarker in postsurgery and other gastrointestinal tumor plasma samples indicated its GC specificity. CONCLUSIONS: EV-derived GClnc1 serves as a circulating biomarker for the early detection of GC, thus providing opportunities for curative surgery and improved survival outcomes.


Asunto(s)
Gastritis Atrófica , Neoplasias Gástricas , Humanos , Biomarcadores de Tumor/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/cirugía , Gastritis Atrófica/diagnóstico , Gastritis Atrófica/genética , Antígeno CA-19-9 , Detección Precoz del Cáncer , Metaplasia
20.
Gastroenterology ; 165(3): 746-761.e16, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37263311

RESUMEN

BACKGROUND & AIMS: Liver fibrosis is an intrinsic wound-healing response to chronic injury and the major cause of liver-related morbidity and mortality worldwide. However, no effective diagnostic or therapeutic strategies are available, owing to its poorly characterized molecular etiology. We aimed to elucidate the mechanisms underlying liver fibrogenesis. METHODS: We performed a quantitative proteomic analysis of clinical fibrotic liver samples to identify dysregulated proteins. Further analyses were performed on the sera of 164 patients with liver fibrosis. Two fibrosis mouse models and several biochemical experiments were used to elucidate liver fibrogenesis. RESULTS: We identified cathepsin S (CTSS) up-regulation as a central node for extracellular matrix remodeling in the human fibrotic liver by proteomic screening. Increased serum CTSS levels efficiently predicted liver fibrosis, even at an early stage. Secreted CTSS cleaved collagen 18A1 at its C-terminus, releasing endostatin peptide, which directly bound to and activated hepatic stellate cells via integrin α5ß1 signaling, whereas genetic ablation of Ctss remarkably suppressed liver fibrogenesis via endostatin reduction in vivo. Further studies identified macrophages as the main source of hepatic CTSS, and splenectomy effectively attenuated macrophage infiltration and CTSS expression in the fibrotic liver. Pharmacologic inhibition of CTSS ameliorated liver fibrosis progression in the mouse models. CONCLUSIONS: CTSS functions as a novel profibrotic factor by remodeling extracellular matrix proteins and may represent a promising target for the diagnosis and treatment of liver fibrosis.


Asunto(s)
Endostatinas , Proteómica , Ratones , Animales , Humanos , Endostatinas/metabolismo , Endostatinas/farmacología , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Fibrosis , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/metabolismo , Matriz Extracelular , Macrófagos/metabolismo
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