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1.
Nat Cancer ; 4(5): 629-647, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37217651

RESUMEN

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Presentación de Antígeno , Neoplasias Pulmonares/patología , Medicina de Precisión , Complejo de la Endopetidasa Proteasomal/metabolismo , Microambiente Tumoral
2.
Mol Cell ; 82(1): 106-122.e9, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34875212

RESUMEN

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.


Asunto(s)
Blastocisto/metabolismo , Linaje de la Célula , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Blastocisto/citología , Cromatina/genética , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Histonas/genética , Humanos , Ratones , Fenotipo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitinas/genética , Ubiquitinas/metabolismo
3.
J Exp Med ; 218(11)2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34477806

RESUMEN

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.


Asunto(s)
Homeostasis/genética , Mutación/genética , Factores de Transcripción/genética , Animales , Autoinmunidad/genética , Cromatina/genética , Disección/métodos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Modelos Animales , Proteína AIRE
4.
J Mol Biol ; 433(21): 167219, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34464654

RESUMEN

Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. Our analysis uncovered thousands of proteins containing such sites, which we term Sites of Alternative Modification (SAMs). Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. SAM-containing proteins are associated with diverse biological functions including cell cycle, DNA damage, and transcriptional regulation. As such, our analysis highlights numerous proteins and pathways as putative targets for further elucidating the crosstalk between ubiquitin and SUMO. Comparing the biological and biochemical properties of SAMs versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Lastly, using S. cerevisiae as model, we show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance.


Asunto(s)
Ciclo Celular/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina/metabolismo , Secuencias de Aminoácidos , Biología Computacional/métodos , Daño del ADN , Humanos , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transcripción Genética , Ubiquitina/genética
5.
ACS Infect Dis ; 7(2): 377-389, 2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33471513

RESUMEN

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.


Asunto(s)
Antifúngicos , Fluconazol , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Colistina/farmacología , Fluconazol/farmacología , Hongos , Permeabilidad
6.
Nat Commun ; 11(1): 409, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31964869

RESUMEN

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.


Asunto(s)
Aparato de Golgi/metabolismo , Ionóforos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis/fisiología , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/metabolismo , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Aparato de Golgi/efectos de los fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Monensina/farmacología , Monensina/uso terapéutico , Mieloma Múltiple/patología , Proteolisis/efectos de los fármacos , Proteostasis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Proteína que Contiene Valosina/metabolismo
7.
Nat Biotechnol ; 2018 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-30346940

RESUMEN

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

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