RESUMEN
Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By using a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle-dependent DSB repair. We show that SCFcyclin F-mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.
Asunto(s)
Ciclo Celular , Ciclinas , Roturas del ADN de Doble Cadena , Reparación del ADN , Exodesoxirribonucleasas , Humanos , Ciclo Celular/genética , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Ciclinas/metabolismo , Ciclinas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Reparación del ADN por Unión de Extremidades , Ubiquitinación , Radiación IonizanteRESUMEN
Sepsis, the dysregulated host response to infection causing life-threatening organ dysfunction, is a global health challenge requiring better understanding of pathophysiology and new therapeutic approaches. Here, we applied high-throughput tandem mass spectrometry to delineate the plasma proteome for sepsis and comparator groups (noninfected critical illness, postoperative inflammation, and healthy volunteers) involving 2612 samples (from 1611 patients) and 4553 liquid chromatography-mass spectrometry analyses acquired through a single batch of continuous measurements, with a throughput of 100 samples per day. We show how this scale of data can delineate proteins, pathways, and coexpression modules in sepsis and be integrated with paired leukocyte transcriptomic data (837 samples from n = 649 patients). We mapped the plasma proteomic landscape of the host response in sepsis, including changes over time, and identified features relating to etiology, clinical phenotypes (including organ failures), and severity. This work reveals subphenotypes informative for sepsis response state, disease processes, and outcome; identifies potential biomarkers; and advances opportunities for a precision medicine approach to sepsis.
Asunto(s)
Proteoma , Sepsis , Humanos , Sepsis/sangre , Proteoma/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteómica/métodos , Masculino , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/análisis , Femenino , Persona de Mediana Edad , Espectrometría de Masas en Tándem/métodosRESUMEN
Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.
Asunto(s)
Antígenos CD , Apirasa , Cadenas alfa de Integrinas , Receptores de Antígenos de Linfocitos T , Transducción de Señal , Animales , Humanos , Ratones , Antígenos CD/metabolismo , Antígenos CD/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Cadenas alfa de Integrinas/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
DNA sensing is important for antiviral immunity. The DNA sensor cGAS synthesizes 2'3'-cyclic GMP-AMP (cGAMP), a second messenger that activates STING, which induces innate immunity. cGAMP not only activates STING in the cell where it is produced but cGAMP also transfers to other cells. Transporters, channels, and pores (including SLC19A1, SLC46A2, P2X7, ABCC1, and volume-regulated anion channels (VRACs)) release cGAMP into the extracellular space and/or import cGAMP. We report that infection with multiple human viruses depletes some of these cGAMP conduits. This includes herpes simplex virus 1 (HSV-1) that targets SLC46A2, P2X7, and the VRAC subunits LRRC8A and LRRC8C for degradation. The HSV-1 protein UL56 is necessary and sufficient for these effects that are mediated at least partially by proteasomal turnover. UL56 thereby inhibits cGAMP uptake via VRAC, SLC46A2, and P2X7. Taken together, HSV-1 antagonizes intercellular cGAMP transfer. We propose that this limits innate immunity by reducing cell-to-cell communication via the immunotransmitter cGAMP.
Asunto(s)
Herpesvirus Humano 1 , Nucleótidos Cíclicos , Animales , Humanos , Células HEK293 , Herpes Simple/virología , Herpes Simple/metabolismo , Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Nucleótidos Cíclicos/metabolismo , Proteínas Virales/metabolismoRESUMEN
Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1ß (IL-1ß) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1ß cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1ß production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.
Asunto(s)
Inflamasomas , Interleucina-1beta , Macrófagos , Microglía , Proteína con Dominio Pirina 3 de la Familia NLR , Proteómica , Ubiquitina Tiolesterasa , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteómica/métodos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Dysregulation of the ubiquitin-proteasome systems is a hallmark of various disease states including neurodegenerative diseases and cancer. Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is expressed primarily in the central nervous system under normal physiological conditions, however, is considered an oncogene in various cancers, including melanoma, lung, breast, and lymphoma. Thus, UCHL1 inhibitors could serve as a viable treatment strategy against these aggressive cancers. Herein, we describe a covalent fragment screen that identified the chloroacetohydrazide scaffold as a covalent UCHL1 inhibitor. Subsequent optimization provided an improved fragment with single-digit micromolar potency against UCHL1 and selectivity over the closely related UCHL3. The molecule demonstrated efficacy in cellular assays of metastasis. Additionally, we report a ligand-bound crystal structure of the most potent molecule in complex with UCHL1, providing insight into the binding mode and information for future optimization.
Asunto(s)
Neoplasias , Ubiquitina Tiolesterasa , Humanos , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Mama , Complejo de la Endopetidasa ProteasomalRESUMEN
KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to â¼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.
Asunto(s)
Neoplasias Colorrectales , Proteínas Proto-Oncogénicas p21(ras) , Ubiquitina-Proteína Ligasas , Humanos , Proteínas Cullin/genética , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/genética , Factores de TranscripciónRESUMEN
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.
Asunto(s)
Proteínas de Unión al ADN , Recombinasas , Humanos , ADN/genética , Reparación del ADN , Replicación del ADN , ADN Cruciforme , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinasas/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.