RESUMO
The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células A549 , Animais , Criança , Pré-Escolar , Feminino , Mutação com Ganho de Função/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Células-Tronco Embrionárias Murinas , Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais , Domínios de Homologia de src/genéticaRESUMO
NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.
Assuntos
Neoplasias , Proteínas de Fusão Oncogênica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Neoplasias/genética , Neoplasias/metabolismo , Fusão Gênica , Receptor trkA/genética , Receptor trkA/metabolismo , Inibidores de Proteínas QuinasesRESUMO
Patients with castration-resistant prostate cancer inevitably acquire resistance to antiandrogen therapies in part because of androgen receptor (AR) mutations or splice variants enabling restored AR signaling. Here we show that ligand-activated AR can form transcriptionally active condensates. Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role. AR liquid-liquid phase separation behaviors faithfully report transcriptional activity and antiandrogen efficacy. Antiandrogens can promote phase separation and transcriptional activity of AR-resistant mutants in a ligand-independent manner. We conducted a phase-separation-based phenotypic screen and identified ET516 that specifically disrupts AR condensates, effectively suppresses AR transcriptional activity and inhibits the proliferation and tumor growth of prostate cancer cells expressing AR-resistant mutants. Our results demonstrate liquid-liquid phase separation as an emerging mechanism underlying drug resistance and show that targeting phase separation may provide a feasible approach for drug discovery.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Receptores Androgênicos/genética , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Ligantes , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
SHP1 is a non-receptor protein tyrosine phosphatase that is widely expressed in hematopoietic cells such as white blood cells, neutrophils, and immune cells. SHP1 can regulate the occurrence and differentiation of immune cells and plays an important role as a tumor suppressor. Previous studies have suggested that SHP2, the homologous protein of phosphatase SHP1, can undergo liquid-liquid phase separation (LLPS). Therefore, in this study, we investigated if SHP1 is also capable of LLPS. To the best of our knowledge, our study is the first to reveal that SHP1 has the ability to undergo LLPS. In addition, we identified an important residue, SHP1-R360E, that can completely inhibit the LLPS ability of SHP1, but this mutation has no remarkable effect on SHP1's enzymatic activity. This allows us to explore the phosphatase activity and phase separation ability of SHP1 separately, providing a basis for future exploration of the phase separation mechanism of phosphatases.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 11 , Diferenciação Celular , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismoRESUMO
Biomolecular condensates play a significant role in chromatin activities, primarily by concentrating and compartmentalizing proteins and/or nucleic acids. However, their genomic landscapes and compositions remain largely unexplored due to a lack of dedicated computational tools for systematic identification in vivo. To address this, we develop CondSigDetector, a computational framework designed to detect condensate-like chromatin-associated protein co-occupancy signatures (CondSigs), to predict genomic loci and component proteins of distinct chromatin-associated biomolecular condensates. Applying this framework to mouse embryonic stem cells (mESC) and human K562 cells enable us to depict the high-resolution genomic landscape of chromatin-associated biomolecular condensates, and uncover both known and potentially unknown biomolecular condensates. Multi-omics analysis and experimental validation further verify the condensation properties of CondSigs. Additionally, our investigation sheds light on the impact of chromatin-associated biomolecular condensates on chromatin activities. Collectively, CondSigDetector provides an approach to decode the genomic landscape of chromatin-associated condensates, facilitating a deeper understanding of their biological functions and underlying mechanisms in cells.
Assuntos
Condensados Biomoleculares , Cromatina , Cromatina/metabolismo , Cromatina/genética , Humanos , Animais , Camundongos , Células K562 , Condensados Biomoleculares/metabolismo , Genômica/métodos , Células-Tronco Embrionárias Murinas/metabolismo , Biologia Computacional/métodos , GenomaRESUMO
Importance: Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to antiseizure medications. While most patients with MTLE do not have pathogenic germline genetic variants, the contribution of postzygotic (ie, somatic) variants in the brain is unknown. Objective: To test the association between pathogenic somatic variants in the hippocampus and MTLE. Design, Setting, and Participants: This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically treated patients with MTLE and age-matched and sex-matched neurotypical controls. Participants treated at level 4 epilepsy centers were enrolled from 1988 through 2019, and clinical data were collected retrospectively. Whole-exome and gene-panel sequencing (each genomic region sequenced more than 500 times on average) were used to identify candidate pathogenic somatic variants. A subset of novel variants was functionally evaluated using cellular and molecular assays. Patients with nonlesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE who underwent anterior medial temporal lobectomy were eligible. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks. Data were analyzed from June 2020 to August 2022. Exposures: Drug-resistant MTLE. Main Outcomes and Measures: Presence and abundance of pathogenic somatic variants in the hippocampus vs the unaffected temporal neocortex. Results: Of 105 included patients with MTLE, 53 (50.5%) were female, and the median (IQR) age was 32 (26-44) years; of 30 neurotypical controls, 11 (36.7%) were female, and the median (IQR) age was 37 (18-53) years. Eleven pathogenic somatic variants enriched in the hippocampus relative to the unaffected temporal neocortex (median [IQR] variant allele frequency, 1.92 [1.5-2.7] vs 0.3 [0-0.9]; P = .01) were detected in patients with MTLE but not in controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF, and NF1, all predicted to constitutively activate Ras/Raf/mitogen-activated protein kinase (MAPK) signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for the PTPN11 variants as a possible dominant gain-of-function mechanism. Conclusions and Relevance: Hippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.
Assuntos
Epilepsia Resistente a Medicamentos , Epilepsia do Lobo Temporal , Epilepsia , Neocórtex , Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Masculino , Epilepsia do Lobo Temporal/cirurgia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estudos Retrospectivos , Hipocampo/patologia , Epilepsia/patologiaRESUMO
OBJECTIVE: To observe effects of Dan Wei Powder (Powder for treating the gall bladder and stomach) Tea Bag (DWSTB) on the aggregation rate of blood platelet in vivo and in vitro. METHODS: Increase of the platelet aggregation rate in the rat in vivo was induced by carrageenin, and increase of the rabbit platelet aggregation rate in vitro was induced by adenosine diphosphate (ADP) and collagen, respectively. The effects of DWSTB on the platelet aggregation rate were investigated in vivo and in vitro, respectively. RESULTS: The maximum in vivo platelet aggregation rate in the rat was significantly decreased after administration of 2.0 and 4.0 g x kg(-1) DWSTB (P < 0.05, P < 0.01). The maximum rabbit platelet aggregation rate induced by ADP and collagen in vitro were suppressed significantly by 2.0-16.0 mg x mL(-1) and 2.0-8.0 mg x mL(-1) DWSTB, respectively (P < 0.05, P < 0.01). And the effect of DWSTB on platelet aggregation was raised with increase of its dose. CONCLUSION: Dan Wei Powder Tea Bag can restrain the aggregation of platelet in vivo and in vitro.
Assuntos
Plaquetas/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/fisiologia , Masculino , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
This paper evaluates the potential usage of graphene/crosslinked polyethylene (graphene/XLPE) as the insulating material for high voltage direct current (HVDC) cables. Thermal, mechanical and electrical properties of blends with/without graphene were evaluated by differential scanning calorimetry (DSC), tensile strength, DC conductivity, space charge measurements and water tree aging test. The results indicate that 0.007-0.008% weight amount of graphene can improve the mechanical and electrical insulation properties of XLPE blends, namely higher tensile/yield strength, improved space charge distribution, and shorter/fewer water tree branches. The improvements mainly attribute to the high stiffness of graphene, deep traps introduced by the interaction zones of graphene and XLPE, and the blockage effect of graphene within XLPE. For thermal performance of XLPE blends, graphene nano-fillers have but limited improvement. The crystallinity of the blends barely changes with the addition of graphene. However, the crosslinking degree increases as the additive-like amounts of graphene doped. The above findings provide a guide for tailoring lightweight XLPE materials with excellent mechanical and electrical performances by doping them with a small amount of graphene.
RESUMO
Tumor metastasis accounts for the majority of cancer-related deaths; it is therefore important to develop preclinical models that faithfully recapitulate disease progression. Here, we generated paired organoids derived from primary tumors and matched liver metastases in the same colorectal cancer (CRC) patients. Despite the fact that paired organoids exhibit comparable gene expression and cell morphology, organoids from metastatic lesions demonstrate more aggressive phenotypes, tumorigenesis, and metastatic capacity than those from primary lesions. Transcriptional analyses of the paired organoids reveal signature genes and pathways altered during the progression of CRC, including SOX2. Further study shows that inducible knockdown of SOX2 attenuated invasion, proliferation, and liver metastasis outgrowth. Taken together, we use patient-derived paired primary and metastatic cancer organoids to model CRC metastasis and illustrate that SOX2 is associated with CRC progression and may serve as a potential prognostic biomarker and therapeutic target of CRC.
Assuntos
Carcinoma/secundário , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Modelos Biológicos , Invasividade Neoplásica/patologia , Organoides , Animais , Biomarcadores Tumorais , Carcinoma/patologia , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Organoides/efeitos dos fármacos , Organoides/patologia , Organoides/transplante , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/fisiologiaRESUMO
Isocitrate dehydrogenase 1 (IDH1), which catalyzes the conversion of isocitrate to α-ketoglutarate, is one of key enzymes in the tricarboxylic acid cycle (TCA). Hotspot mutation at Arg132 in IDH1 that alters the function of IDH1 by further converting the α-ketoglutarate(α-KG) to 2-hydroxyglutarate (2-HG) have been identified in a variety of cancers. Because the IDH1 mutations occur in a significant portion of gliomas and glioblastomas, it is important that IDH1 inhibitors have to be brain penetrant to treat IDH1-mutant brain tumors. Here we report the efforts to design and synthesize a novel serial of mutant IDH1 inhibitors with improved activity and the blood-brain barrier (BBB) penetration. We show that compound 5 exhibits good brain exposure and potent 2-HG inhibition in a HT1080-derived mouse xenograft model, which makes it a potential preclinical candidate to treat IDH1-mutant brain tumors.