RESUMO
Rexinoids are compounds that bind to the rexinoid X receptor (RXR) to modulate gene expression and have been proposed as a new class of therapeutics to treat Alzheimer's disease. Different rexinoids will initiate downstream effects that can be quite marked even though such compounds can be structurally similar and have comparable RXR binding affinities. RXR can both homo- and heterodimerize, and these protein-protein interactions and subsequent transactivating potential lead to differential gene expression, depending on the RXR dimeric partner, additional cofactors recruited, and downstream transcription factors that are up- or downregulated. Expression analysis was performed in the U87 human glioblastoma cell line treated with a panel of rexinoids, and our analysis demonstrated that rexinoids with similar RXR EC50 values can have pronounced differences in differential gene expression. Rexinoid binding likely leads to distinctive RXR conformations that cause major downstream gene expression alterations via modulation of RXR interacting proteins. Yeast two-hybrid analysis of RXR bait with two RXR interacting partners demonstrates that rexinoids drive differential binding of RXR to distinctive protein partners. Physiochemical analysis of the rexinoids reveals that the molecules cluster similarly to their gene expression patterns. Thus, rexinoids with similar RXR binding affinities drive differential gene expression by stimulating additional binding patterns in RXR and its homo- and heteropartners, driven by the physicochemical characteristics of these molecules.
Assuntos
Glioblastoma , Receptores X de Retinoides , Técnicas do Sistema de Duplo-Híbrido , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Linhagem Celular Tumoral , Receptores X de Retinoides/metabolismo , Receptores X de Retinoides/genética , Ligação Proteica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
The programmed cell death protein 1 (PD-1) plays a critical role in cancer immune evasion. Blocking the PD-1-PD-L1 interaction by monoclonal antibodies has shown remarkable clinical efficacy in treating certain types of cancer. However, antibodies are costly to produce, and antibody-based therapies can cause immune-related adverse events. To address the limitations associated with current PD-1/PD-L1 blockade immunotherapy, we aimed to develop peptide-based inhibitors of the PD-1/PD-L1 interaction as an alternative means to PD-1/PD-L1 blockade antibodies for anti-cancer immunotherapy. Through the functional screening of peptide arrays encompassing the ectodomains of PD-1 and PD-L1, followed by the optimization of the hit peptides for solubility and stability, we have identified a 16-mer peptide, named mL7N, with a remarkable efficacy in blocking the PD-1/PD-L1 interaction both in vitro and in vivo. The mL7N peptide effectively rejuvenated PD-1-suppressed T cells in multiple cellular systems designed to recapitulate the PD-1/PD-L1 interaction in the context of T-cell receptor signaling. Furthermore, PA-mL7N, a chimera of the mL7N peptide coupled to albumin-binding palmitic acid (PA), significantly promoted breast cancer cell killing by peripheral blood mononuclear cells ex vivo and significantly curbed tumor growth in a syngeneic mouse model of breast cancer. Our work raises the prospect that mL7N may serve as a prototype for the development of a new line of peptide-based immunomodulators targeting the PD-1/PD-L1 immune checkpoint with potential applications in cancer treatment.
Assuntos
Antígeno B7-H1 , Peptídeos , Receptor de Morte Celular Programada 1 , Linfócitos T , Animais , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Camundongos , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Peptídeos/farmacologia , Peptídeos/química , Feminino , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Linhagem Celular Tumoral , Ligação Proteica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacosRESUMO
BACKGROUND: Drug resistance, including Adriamycin-based therapeutic resistance, remains a challenge in breast cancer (BC) treatment. Studies have revealed that macrophages could play a pivotal role in mediating the chemoresistance of cancer cells. Accumulating evidence suggests that tRNA-Derived small RNAs (tDRs) are associated the physiological and pathological processes in multiple cancers. However, the underlying mechanisms of tDRs on chemoresistance of BC in tumor-associated macrophages remain largely unknown. METHODS: The high-throughput sequencing technique was used to screen tDRs expression profile in BC cells. Gain- and loss-of-function experiments and xenograft models were performed to verify the biological function of 3'tRF-Ala-AGC in BC cells. The CIBERSORT algorithm was used to investigate immune cell infiltration in BC tissues. To explore the role of 3'tRF-Ala-AGC in macrophages, M2 macrophages transfected with 3'tRF-Ala-AGC mimic or inhibitor were co-cultured with BC cells. Effects on Nuclear factor-κb (NF-κb) pathway were investigated by NF-κb nuclear translocation assay and western blot analysis. RNA pull-down assay was performed to identify 3'tRF-Ala-AGC interacting proteins. RESULTS: A 3'tRF fragment of 3'tRF-AlaAGC was screened, which is significantly overexpressed in BC specimens and Adriamycin-resistant cells. 3'tRF-AlaAGC could promote cell malignant activity and facilitate M2 polarization of macrophages in vitro and in vivo. Higher expression of M2 macrophages were more likely to have lymph node metastasis and deeper invasion in BC patients. Mechanistically, 3'tRF-AlaAGC binds Type 1-associated death domain protein (TRADD) in BC cells, and suppression of TRADD partially abolished the enhanced effect of 3'tRF-AlaAGC mimic on phenotype of M2. The NF-κb signaling pathway was activated in BC cells co-cultured with M2 macrophages transfected with 3'tRF-AlaAGC mimic. CONCLUSIONS: 3'tRF-AlaAGC might modulate macrophage polarization via binding to TRADD and increase the effect of M2 on promoting the chemoresistance in BC cells through NF-κb signaling pathway.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Macrófagos , NF-kappa B , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Macrófagos/metabolismo , Animais , Linhagem Celular Tumoral , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA de Transferência/metabolismo , RNA de Transferência/genética , Polaridade Celular/efeitos dos fármacos , Camundongos , Transdução de Sinais , Camundongos Nus , Doxorrubicina/farmacologia , Camundongos Endogâmicos BALB CRESUMO
Shoot branching is determined by a balance between factors that promote axillary bud dormancy and factors that release buds from the quiescent state. The TCP family of transcription factors is classified into two classes, Class I and Class II, which usually play different roles. While the role of the Class II TCP BRANCHED1 (BRC1) in suppressing axillary bud development in Arabidopsis thaliana has been widely explored, the function of Class I TCPs in this process remains unknown. We analyzed the role of Class I TCP14 and TCP15 in axillary branch development in Arabidopsis through a series of genetic and molecular studies. In contrast to the increased branch number shown by brc1 mutants, tcp14 tcp15 plants exhibit a reduced number of branches compared with wild-type. Our findings provide evidence that TCP14 and TCP15 act by counteracting BRC1 function through two distinct mechanisms. First, they indirectly reduce BRC1 expression levels. Additionally, TCP15 directly interacts with BRC1 decoying it from chromatin and thereby preventing the transcriptional activation of a set of BRC1-dependent genes. We describe a molecular mechanism by which Class I TCPs physically antagonize the action of the Class II TCP BRC1, aligning with their opposite roles in axillary bud development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Cromatina/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genéticaRESUMO
Cone enlargement is a crucial process for seed production and reproduction in gymnosperms. Most of our knowledge of cone development is derived from observing anatomical structure during gametophyte development. Therefore, the exact molecular mechanism underlying cone enlargement after fertilization is poorly understood. Here, we demonstrate that sucrose promotes cone enlargement in Torreya grandis, a gymnosperm species with relatively low rates of cone enlargement, via the TgNGA1-TgWRKY47-TgEXPA2 pathway. Cell expansion plays a significant role in cone enlargement in T. grandis. 13C labeling and sucrose feeding experiments indicated that sucrose-induced changes in cell size and number contribute to cone enlargement in this species. RNA-sequencing analysis, transient overexpression in T. grandis cones, and stable overexpression in tomato (Solanum lycopersicum) suggested that the expansin gene TgEXPA2 positively regulates cell expansion in T. grandis cones. The WRKY transcription factor TgWRKY47 directly enhances TgEXPA2 expression by binding to its promoter. Additionally, the NGATHA transcription factor TgNGA1 directly interacts with TgWRKY47. This interaction suppresses the DNA-binding ability of TgWRKY47, thereby reducing its transcriptional activation on TgEXPA2 without affecting the transactivation ability of TgWRKY47. Our findings establish a link between sucrose and cone enlargement in T. grandis and elucidate the potential underlying molecular mechanism.
Assuntos
Proteínas de Plantas , Sacarose , Taxaceae , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Sacarose/metabolismo , Sacarose/farmacologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Taxaceae/genética , Taxaceae/crescimento & desenvolvimentoRESUMO
Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.
Assuntos
Ciclopentanos , Regulação da Expressão Gênica de Plantas , Giberelinas , Malus , Oxilipinas , Proteínas de Plantas , Transdução de Sinais , Ubiquitinação , Oxilipinas/metabolismo , Malus/genética , Malus/metabolismo , Ciclopentanos/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Giberelinas/metabolismo , Proteólise/efeitos dos fármacos , Antocianinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Modelos BiológicosRESUMO
Wood is resulted from the radial growth paced by the division and differentiation of vascular cambium cells in woody plants, and phytohormones play important roles in cambium activity. Here, we identified that PagJAZ5, a key negative regulator of jasmonate (JA) signaling, plays important roles in enhancing cambium cell division and differentiation by mediating cytokinin signaling in poplar 84K (Populus alba × Populus glandulosa). PagJAZ5 is preferentially expressed in developing phloem and cambium, weakly in developing xylem cells. Overexpression (OE) of PagJAZ5m (insensitive to JA) increased cambium activity and xylem differentiation, while jaz mutants showed opposite results. Transcriptome analyses revealed that cytokinin oxidase/dehydrogenase (CKXs) and type-A response regulators (RRs) were downregulated in PagJAZ5m OE plants. The bioactive cytokinins were significantly increased in PagJAZ5m overexpressing plants and decreased in jaz5 mutants, compared with that in 84K plants. The PagJAZ5 directly interact with PagMYC2a/b and PagWOX4b. Further, we found that the PagRR5 is regulated by PagMYC2a and PagWOX4b and involved in the regulation of xylem development. Our results showed that PagJAZ5 can increase cambium activity and promote xylem differentiation through modulating cytokinin level and type-A RR during wood formation in poplar.
Assuntos
Câmbio , Ciclopentanos , Citocininas , Regulação da Expressão Gênica de Plantas , Oxilipinas , Proteínas de Plantas , Populus , Transdução de Sinais , Xilema , Populus/genética , Populus/crescimento & desenvolvimento , Populus/metabolismo , Câmbio/genética , Câmbio/crescimento & desenvolvimento , Câmbio/metabolismo , Citocininas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Xilema/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Diferenciação CelularRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA , Proteína 1 Homóloga a MutL , Ligação Proteica , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Ligação Proteica/efeitos dos fármacos , Linhagem Celular Tumoral , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Neurônios/metabolismo , Pessoa de Meia-Idade , MasculinoRESUMO
Intervertebral disc degeneration (IVDD) is a common degenerative disease which is closely related to low back pain (LBP) and brings huge economic and social burdens. In this study, we explored the therapeutic effects of Homoplantaginin (Hom) for IVDD due to its convincing anti-inflammatory and antioxidant functions. TNF-α was used to simulate the inflammatory environment for nucleus pulposus (NP) cells in vitro. We verified that Hom could alleviate the TNF-α-induced inflammation and disturbance of ECM homeostasis through blocking the NF-κB/MAPK signaling pathways. Subsequently, we screened the binding targets of Hom and confirmed that Hom could directly bind to TAK1 and inhibit its phosphorylation to down-regulate the inflammation-related pathways. The therapeutic effects of Hom on IVDD were further validated through a needle puncture rat model in vivo. Overall, Hom was a promising small molecule for IVDD early intervention, possessing huge clinical translational value.
Assuntos
Degeneração do Disco Intervertebral , MAP Quinase Quinase Quinases , NF-kappa B , Animais , Humanos , Masculino , Ratos , Células Cultivadas , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Núcleo Pulposo/metabolismo , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/patologia , Ligação Proteica/fisiologia , Ligação Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Thymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein-protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.
Assuntos
Citocinas , Receptores de Citocinas , Linfopoietina do Estroma do Timo , Humanos , Citocinas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Citocinas/antagonistas & inibidores , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , Células Th2/imunologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Animais , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Ligação Proteica/efeitos dos fármacos , Interleucina-4/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Simulação de Acoplamento MolecularRESUMO
In flowering plants, sexual reproductive success depends on the production of viable pollen grains. However, the mechanisms by which QUA QUINE STARCH (QQS) regulates pollen development and how transcriptional activators facilitate the transcription of QQS in this process remain poorly understood. Here, we demonstrate that INDUCER OF CBF EXPRESSION 1 (ICE1), a basic helix-loop-helix (bHLH) transcription factor, acts as a key transcriptional activator and positively regulates QQS expression to increase pollen germination and viability in Arabidopsis thaliana by interacting with INDETERMINATE DOMAIN14 (IDD14). In our genetic and biochemical experiments, overexpression of ICE1 greatly promoted both the activation of QQS and high pollen viability mediated by QQS. IDD14 additively enhanced ICE1 function by promoting the binding of ICE1 to the QQS promoter. In addition, mutation of ICE1 significantly repressed QQS expression; the impaired function of QQS and the abnormal anther dehiscence jointly affected pollen development of the ice1-2 mutant. Our results also showed that the enhancement of pollen activity by ICE1 depends on QQS. Furthermore, QQS interacted with CUT1, the key enzyme for long-chain lipid biosynthesis. This interaction both promoted CUT1 activity and regulated pollen lipid metabolism, ultimately determining pollen hydration and fertility. Our results not only provide new insights into the key function of QQS in promoting pollen development by regulating pollen lipid metabolism, but also elucidate the mechanism that facilitates the transcription of QQS in this vital developmental process.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Pólen , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Pólen/crescimento & desenvolvimento , Pólen/genética , Pólen/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Germinação/genética , Germinação/efeitos dos fármacos , Amido/metabolismo , Ligação Proteica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Fatores de TranscriçãoRESUMO
OBJECTIVE: We have previously shown that the anti-cancer peptide PNC-27 kills cancer cells by co-localizing with membrane-expressed HDM-2, resulting in transmembrane pore formation causing extrusion of intracellular contents. We have also observed cancer cell mitochondrial disruption in PNC-27-treated cancer cells. Our objectives are to determine: 1. if PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) in the cancer cell membrane and 2. if this peptide causes selective disruption of cancer cell mitochondria. METHODS: For aim 1, we incubated MIA-PaCa-2 human pancreatic carcinoma cells with PNC-27 in the presence of a monoclonal antibody against the amino terminal p53 binding site of HDM-2 to determine if it, but not negative control immune serum, blocks PNC-27-induced tumor cell necrosis. For the second aim, we incubated these cells with PNC-27 in the presence of two specific dyes that highlight normal organelle function: mitotracker for mitochondria and lysotracker for lysosomes. We also performed immuno-electron microscopy (IEM) with gold-labeled anti-PNC-27 antibody on the mitochondria of these cells treated with PNC-27. RESULTS: Monoclonal antibody to the p53 binding site of HDM-2 blocks PNC-27-induced cancer cell necrosis, whereas negative control immune serum does not. The mitochondria of PNC-27-treated cancer cells fail to retain mitotracker dye while their lysosomes retain lysotracker dye. IEM of the mitochondria cancer cells reveals gold particles present on the mitochondrial membranes. CONCLUSIONS: PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) inducing transmembrane pore formation and cancer cell necrosis. Furthermore, this peptide enters cancer cells and binds to the membranes of mitochondria, resulting in their disruption.
Assuntos
Membrana Celular , Membranas Mitocondriais , Proteínas Proto-Oncogênicas c-mdm2 , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/metabolismo , NecroseRESUMO
Water plays crucial roles in expeditious growth and osmotic stress of bamboo. Nevertheless, the molecular mechanism of water transport remains unclear. In this study, an aquaporin gene, PeTIP4-3, was identified through a joint analysis of root pressure and transcriptomic data in moso bamboo (Phyllostachys edulis). PeTIP4-3 was highly expressed in shoots, especially in the vascular bundle sheath cells. Overexpression of PeTIP4-3 could increase drought and salt tolerance in transgenic yeast and rice. A co-expression pattern of PeSAPK4, PeMYB99 and PeTIP4-3 was revealed by WGCNA. PeMYB99 exhibited an ability to independently bind to and activate PeTIP4-3, which augmented tolerance to drought and salt stress. PeSAPK4 could interact with and phosphorylate PeMYB99 in vivo and in vitro, wherein they synergistically accelerated PeTIP4-3 transcription. Overexpression of PeMYB99 and PeSAPK4 also conferred drought and salt tolerance in transgenic rice. Further ABA treatment analysis indicated that PeSAPK4 enhanced water transport in response to stress via ABA signaling. Collectively, an ABA-mediated cascade of PeSAPK4-PeMYB99-PeTIP4-3 is proposed, which governs water transport in moso bamboo.
Assuntos
Aquaporinas , Secas , Regulação da Expressão Gênica de Plantas , Oryza , Proteínas de Plantas , Plantas Geneticamente Modificadas , Água , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Água/metabolismo , Oryza/genética , Oryza/metabolismo , Oryza/fisiologia , Aquaporinas/metabolismo , Aquaporinas/genética , Transporte Biológico , Poaceae/genética , Poaceae/fisiologia , Modelos Biológicos , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Tolerância ao Sal/genética , Fosforilação , Ligação Proteica/efeitos dos fármacos , Estresse FisiológicoRESUMO
Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes.
Assuntos
Ciclopentanos , Regulação da Expressão Gênica de Plantas , Oryza , Oxilipinas , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas , Rhizoctonia , Ácido Salicílico , Xanthomonas , Oxilipinas/metabolismo , Ácido Salicílico/metabolismo , Ciclopentanos/metabolismo , Oryza/microbiologia , Oryza/genética , Oryza/imunologia , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Xanthomonas/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Rhizoctonia/fisiologia , Imunidade Vegetal/efeitos dos fármacos , Mutação/genética , Resistência à Doença/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica/efeitos dos fármacosRESUMO
Follicular maturation arrest is a prevalent endocrine disorder characterized by hormonal imbalance, ovarian dysfunction, and metabolic disturbances leading to Polycystic ovarian syndrome (PCOS). Tanshinone IIA (TIIA), a bioactive compound derived from Salvia miltiorrhiza, has shown promising therapeutic potential in various diseases, including cardiovascular diseases and cancer. However, its effects on reproductive health and gynecological disorders, particularly PCOS, remain poorly understood. In this study, we investigated the potential therapeutic effects of TIIA on ovarian function. Using a combination of experimental and computational approaches, we elucidated the molecular mechanisms underlying TIIA's pharmacological impact on ovarian function, follicular development, and androgen receptor signaling. Molecular docking and dynamics simulations revealed that TIIA interacts with the human androgen receptor (HAR), modulating its activity and downstream signaling pathways. Our results demonstrate that TIIA treatment alleviates PCOS-like symptoms in a zebrafish model, including improved follicular development, lowered GSI index, improved antioxidant status (SOD, CAT), decreased LDH levels, and enhanced AChE levels by regulating Tox3 and Dennd1a pathway. Our findings suggest that TIIA may hold promise as a novel therapeutic agent for the management of PCOS or ovulation induction.
Assuntos
Abietanos , Folículo Ovariano , Síndrome do Ovário Policístico , Receptores Androgênicos , Salvia miltiorrhiza , Peixe-Zebra , Animais , Humanos , Abietanos/farmacologia , Receptores Androgênicos/metabolismo , Salvia miltiorrhiza/química , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Folículo Ovariano/patologia , Feminino , Simulação de Acoplamento Molecular , Proteínas de Peixe-Zebra/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.
Assuntos
Movimento Celular , Estetrol , Regulação Neoplásica da Expressão Gênica , Inibidor 2 de Ativador de Plasminogênio , Receptores Acoplados a Proteínas G , Transdução de Sinais , Neoplasias de Mama Triplo Negativas , Feminino , Humanos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Estetrol/farmacologia , Estetrol/metabolismo , Invasividade Neoplásica , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Calcium oscillations are induced by different stresses. Calcium-dependent protein kinases (CDPKs/CPKs) are one major group of the plant calcium decoders that are involved in various processes including drought response. Some CPKs are calcium-independent. Here, we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool. We found that ZmCPK2 does not bind calcium, and its activity is mainly inhibited during short term abscisic acid (ABA) treatment, and dynamically changed in prolonged treatment. Interestingly, ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17, a positive regulator of drought resistance, which is activated by ABA. ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60. ZmCPK2 interacts with and phosphorylates and activates ZmYAB15, a negative transcriptional factor for drought resistance. Our results suggest that drought stress-induced Ca2+ can be decoded directly by ZmCPK17 that inhibits ZmCPK2, thereby promoting plant adaptation to water deficit.
Assuntos
Ácido Abscísico , Cálcio , Secas , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Proteínas Quinases , Zea mays , Zea mays/efeitos dos fármacos , Zea mays/metabolismo , Zea mays/genética , Zea mays/fisiologia , Cálcio/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fosforilação , Proteínas Quinases/metabolismo , Plantas Geneticamente Modificadas , Estresse Fisiológico/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions. .
Assuntos
Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2 , Tretinoína , Proteína de Morte Celular Associada a bcl , Animais , Masculino , Ratos , Apoptose/efeitos dos fármacos , Proteína de Morte Celular Associada a bcl/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/patologia , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.
Assuntos
Movimento Celular , Proliferação de Células , Receptores de Interleucina-6 , Humanos , Proliferação de Células/efeitos dos fármacos , Receptores de Interleucina-6/metabolismo , Movimento Celular/efeitos dos fármacos , Células HEK293 , Linhagem Celular Tumoral , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: Major depressive disorder (MDD) is commonly treated with selective serotonin reuptake inhibitors (SSRIs). SSRIs inhibit the serotonin transporter (5-HTT), but the downstream antidepressant mechanism of action of these drugs is poorly understood. The serotonin 1B (5-HT1B) receptor is functionally linked to 5-HTT and 5-HT1B receptor binding and 5-HT1B receptor mRNA is reduced in the raphe nuclei after SSRI administration in primates and rodents, respectively. The effect of SSRI treatment on 5-HT1B receptor binding in patients with MDD has not been examined previously. This positron emission tomography (PET) study aimed to quantify brain 5-HT1B receptor binding changes in vivo after SSRI treatment for MDD in relation to treatment effect. METHODS: Eight unmedicated patients with moderate to severe MDD underwent PET with the 5-HT1B receptor radioligand [11C]AZ10419369 before and after 3 to 4 weeks of treatment with the SSRI escitalopram 10 mg daily. Depression severity was assessed at time of PET and after 6 to 7 weeks of treatment with the Montgomery-Åsberg Depression Rating Scale. RESULTS: We observed a significant reduction in [11C]AZ10419369 binding in a dorsal brainstem (DBS) region containing the median and dorsal raphe nuclei after escitalopram treatment (P = .036). Change in DBS [11C]AZ10419369 binding correlated with Montgomery-Åsberg Depression Rating Scale reduction after 3-4 (r = 0.78, P = .021) and 6-7 (r = 0.94, P < .001) weeks' treatment. CONCLUSIONS: Our findings align with the previously reported reduction of 5-HT1B receptor binding in the raphe nuclei after SSRI administration and support future studies testing change in DBS 5-HT1B receptor binding as an SSRI treatment response marker.