RESUMO
Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage-induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.
Assuntos
Dano ao DNA , Reparo do DNA , Fatores de Transcrição de Domínio TEA , Humanos , Carcinogênese/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismoRESUMO
Liquid chromatography coupled with bottom-up mass spectrometry (LC-MS/MS)-based proteomics is increasingly used to detect changes in posttranslational modifications (PTMs) in samples from different conditions. Analysis of data from such experiments faces numerous statistical challenges. These include the low abundance of modified proteoforms, the small number of observed peptides that span modification sites, and confounding between changes in the abundance of PTM and the overall changes in the protein abundance. Therefore, statistical approaches for detecting differential PTM abundance must integrate all the available information pertaining to a PTM site and consider all the relevant sources of confounding and variation. In this manuscript, we propose such a statistical framework, which is versatile, accurate, and leads to reproducible results. The framework requires an experimental design, which quantifies, for each sample, both peptides with PTMs and peptides from the same proteins with no modification sites. The proposed framework supports both label-free and tandem mass tag-based LC-MS/MS acquisitions. The statistical methodology separately summarizes the abundances of peptides with and without the modification sites, by fitting separate linear mixed effects models appropriate for the experimental design. Next, model-based inferences regarding the PTM and the protein-level abundances are combined to account for the confounding between these two sources. Evaluations on computer simulations, a spike-in experiment with known ground truth, and three biological experiments with different organisms, modification types, and data acquisition types demonstrate the improved fold change estimation and detection of differential PTM abundance, as compared to currently used approaches. The proposed framework is implemented in the free and open-source R/Bioconductor package MSstatsPTM.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Proteômica/métodos , Cromatografia Líquida , Processamento de Proteína Pós-Traducional , Proteínas , Peptídeos/químicaRESUMO
The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.
Assuntos
Neoplasias , Animais , Humanos , Proteólise , Neoplasias/genética , Mutação , Mamíferos , Fatores de Transcrição/genética , DNA Helicases/genética , Proteínas Nucleares/genéticaRESUMO
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
Assuntos
Carcinogênese/metabolismo , Dimerização , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas ras/genéticaRESUMO
Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.
Assuntos
Homeostase/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Apoptose/fisiologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Chlorocebus aethiops , Feminino , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica/patologia , Proteômica/métodos , Transdução de Sinais/fisiologiaRESUMO
The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.
Assuntos
Anticorpos/química , Peptídeos/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Células Cultivadas , Cristalografia por Raios X/métodos , Humanos , Espectrometria de Massas/métodos , Ligação Proteica , Proteômica/métodos , Coelhos , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/imunologia , UbiquitinaçãoRESUMO
Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.
Assuntos
Proteínas Relacionadas à Autofagia/deficiência , Autofagia , Disenteria Bacilar/microbiologia , Imunidade Inata , Macrófagos/microbiologia , Estresse Oxidativo , Proteoma , Proteômica , Shigella flexneri/patogenicidade , Animais , Proteínas Relacionadas à Autofagia/genética , Células Cultivadas , Modelos Animais de Doenças , Disenteria Bacilar/imunologia , Disenteria Bacilar/metabolismo , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Shigella flexneri/imunologia , Shigella flexneri/metabolismo , VirulênciaRESUMO
Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
Assuntos
Caspase 8/metabolismo , Citocinas/imunologia , Imunidade Inata/imunologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Citocinas/antagonistas & inibidores , Humanos , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In addition to amyloid-ß plaques and tau tangles, mitochondrial dysfunction is implicated in the pathology of Alzheimer's disease (AD). Neurons heavily rely on mitochondrial function, and deficits in brain energy metabolism are detected early in AD; however, direct human genetic evidence for mitochondrial involvement in AD pathogenesis is limited. We analyzed whole-exome sequencing data of 4549 AD cases and 3332 age-matched controls and discovered that rare protein altering variants in the gene pentatricopeptide repeat-containing protein 1 (PTCD1) show a trend for enrichment in cases compared with controls. We show here that PTCD1 is required for normal mitochondrial rRNA levels, proper assembly of the mitochondrial ribosome and hence for mitochondrial translation and assembly of the electron transport chain. Loss of PTCD1 function impairs oxidative phosphorylation and forces cells to rely on glycolysis for energy production. Cells expressing the AD-linked variant of PTCD1 fail to sustain energy production under increased metabolic stress. In neurons, reduced PTCD1 expression leads to lower ATP levels and impacts spontaneous synaptic activity. Thus, our study uncovers a possible link between a protein required for mitochondrial function and energy metabolism and AD risk.SIGNIFICANCE STATEMENT Mitochondria are the main source of cellular energy and mitochondrial dysfunction is implicated in the pathology of Alzheimer's disease (AD) and other neurodegenerative disorders. Here, we identify a variant in the gene PTCD1 that is enriched in AD patients and demonstrate that PTCD1 is required for ATP generation through oxidative phosphorylation. PTCD1 regulates the level of 16S rRNA, the backbone of the mitoribosome, and is essential for mitochondrial translation and assembly of the electron transport chain. Cells expressing the AD-associated variant fail to maintain adequate ATP production during metabolic stress, and reduced PTCD1 activity disrupts neuronal energy homeostasis and dampens spontaneous transmission. Our work provides a mechanistic link between a protein required for mitochondrial function and genetic AD risk.