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1.
EMBO J ; 43(8): 1445-1483, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38499786

RESUMO

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.


Assuntos
Apoferritinas , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Apoferritinas/genética , Apoferritinas/metabolismo , Linhagem da Célula/genética , Citosina/metabolismo , Fatores de Transcrição Forkhead , Ferro/metabolismo
2.
Mol Cell Proteomics ; 19(2): 261-277, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31772060

RESUMO

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered on single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells' response to combinatorial treatment. We here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a likely cause of antitumor activity. We propose that the inhibition of the autophagic flux on combinatorial treatment is attributed to the major cytoskeletal changes induced on ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/farmacologia , Humanos , Imidazóis/farmacologia , Espectrometria de Massas , Oxidiazóis/farmacologia , Proteômica
3.
Commun Biol ; 2: 325, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508500

RESUMO

Extracellular vesicles (EVs) are a potential source of disease-associated biomarkers for diagnosis. In breast cancer, comprehensive analyses of EVs could yield robust and reliable subtype-specific biomarkers that are still critically needed to improve diagnostic routines and clinical outcome. Here, we show that proteome profiles of EVs secreted by different breast cancer cell lines are highly indicative of their respective molecular subtypes, even more so than the proteome changes within the cancer cells. Moreover, we detected molecular evidence for subtype-specific biological processes and molecular pathways, hyperphosphorylated receptors and kinases in connection with the disease, and compiled a set of protein signatures that closely reflect the associated clinical pathophysiology. These unique features revealed in our work, replicated in clinical material, collectively demonstrate the potential of secreted EVs to differentiate between breast cancer subtypes and show the prospect of their use as non-invasive liquid biopsies for diagnosis and management of breast cancer patients.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/ultraestrutura , Bovinos , Linhagem Celular Tumoral , Vesículas Extracelulares/ultraestrutura , Feminino , Humanos , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Análise de Componente Principal , Proteoma/metabolismo
4.
Cell Syst ; 9(4): 366-374.e5, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31521607

RESUMO

Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.


Assuntos
Neoplasias da Mama/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Peptídeos/metabolismo , Proteômica/métodos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Biópsia por Agulha , Ativação Enzimática , Feminino , Humanos , Células Jurkat , Espectrometria de Massas , Fosforilação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/metabolismo
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