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2.
Nat Commun ; 12(1): 1736, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741957

RESUMO

Metastasis is the leading cause of cancer-related death. Despite the recent advancements in cancer treatment, there is currently no approved therapy for metastasis. The present study reveals a potent and selective activity of PRAK in the regulation of tumor metastasis. While showing no apparent effect on the growth of primary breast cancers or subcutaneously inoculated tumor lines, Prak deficiency abrogates lung metastases in PyMT mice or mice receiving intravenous injection of tumor cells. Consistently, PRAK expression is closely associated with metastatic risk in human cancers. Further analysis indicates that loss of function of PRAK leads to a pronounced inhibition of HIF-1α protein synthesis, possibly due to reduced mTORC1 activities. Notably, pharmacological inactivation of PRAK with a clinically relevant inhibitor recapitulates the anti-metastatic effect of Prak depletion, highlighting the therapeutic potential of targeting PRAK in the control of metastasis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/genética
3.
Nat Commun ; 12(1): 1731, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741962

RESUMO

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.


Assuntos
Sobrevivência Celular/fisiologia , Cerebelo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte Proteico/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Exossomos/metabolismo , Feminino , Interneurônios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Mutação , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Canais de Potássio Shaw/genética , Transdução de Sinais
4.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668971

RESUMO

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein-protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.


Assuntos
Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Biblioteca de Peptídeos , Peptídeos Cíclicos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Ligação Proteica
5.
Nat Commun ; 12(1): 1379, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654074

RESUMO

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Inibidor de NF-kappaB alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
6.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(2): 249-251, 2021 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-33729150

RESUMO

The main pathophysiological changes of acute respiratory distress syndrome (ARDS) are massive destruction of pulmonary vascular endothelial barrier, pulmonary edema, infiltration of inflammatory cells, and refractory hypoxemia in severe cases. Pyroptosis is programmed cell necrosis, triggered by caspase and mediated by proteins in a member of conserved protein family Gasdermin D (GSDMD), which manifests as continuous cell expansion until cell membrane rupture, leading to release of cell contents and activation of a strong inflammatory response. Pyroptosis plays a key role in the development of septic ARDS. In this paper, the molecular mechanism of pyroptosis and the related researches on pyroptosis and ARDS are reviewed.


Assuntos
Piroptose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Necrose , Proteínas de Ligação a Fosfato
7.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670044

RESUMO

Kallmann syndrome is the result of innate genetic defects in the fibroblast growth factor (FGF) regulated signaling network causing diminished signal transduction. One of the rare mutations associated with the syndrome alters the Sprouty (Spry)4 protein by converting the serine at position 241 into a tyrosine. In this study, we characterize the tyrosine Spry4 mutant protein in the primary human embryonic lung fibroblasts WI-38 and osteosarcoma-derived cell line U2OS. As demonstrated in a cell signaling assay, Spry4 gains the capability of inhibiting FGF, but not epithelial growth factor (EGF)-induced signaling as a consequence of the tyrosine substitution. Additionally, migration of normal embryonic lung fibroblasts and osteosarcoma-derived cells is potently inhibited by the tyrosine Spry4 variant, while an effect of the wildtype Spry4 protein is hardly measureable. Concerning cell proliferation, the unaltered Spry4 protein is ineffective to influence the WI-38 cells, while the mutated Spry4 protein decelerates the cell doubling. In summary, these data emphasize that like the other mutations associated with Kallmann syndrome the described Spry4 mutation creates a hyperactive version of a selective inhibitory molecule and can thereby contribute to a weakened FGF signaling. Additionally, the study pinpoints a Spry4 variation expanding the applicability of Spry4 in a potential cancer therapy.


Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome de Kallmann/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteossarcoma/patologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Tirosina/metabolismo
8.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671123

RESUMO

The immune system constantly monitors the emergence of cancerous cells and eliminates them. CD8+ cytotoxic T lymphocytes (CTLs), which kill tumor cells and provide antitumor immunity, select their targets by recognizing tumor antigenic peptides presented by MHC class-I (MHC-I) molecules. Cancer cells circumvent immune surveillance using diverse strategies. A key mechanism of cancer immune evasion is downregulation of MHC-I and key proteins of the antigen processing and presentation machinery (APM). Even though impaired MHC-I expression in cancers is well-known, reversing the MHC-I defects remains the least advanced area of tumor immunology. The discoveries that NLRC5 is the key transcriptional activator of MHC-I and APM genes, and genetic lesions and epigenetic modifications of NLRC5 are the most common cause of MHC-I defects in cancers, have raised the hopes for restoring MHC-I expression. Here, we provide an overview of cancer immunity mediated by CD8+ T cells and the functions of NLRC5 in MHC-I antigen presentation pathways. We describe the impressive advances made in understanding the regulation of NLRC5 expression, the data supporting the antitumor functions of NLRC5 and a few reports that argue for a pro-tumorigenic role. Finally, we explore the possible avenues of exploiting NLRC5 for cancer immunotherapy.


Assuntos
Genes MHC Classe I , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Transativadores/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Humanos
9.
Arch Immunol Ther Exp (Warsz) ; 69(1): 6, 2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33683459

RESUMO

The pathophysiology of rotator cuff tendinopathy is not fully understood, particularly in terms of the local inflammatory process. This study aimed to investigate the expression of selected molecules in the tumour necrosis factor (TNF)-α transduction pathway, including TNF-α, TNF receptor 1 (TNFR1), neutral sphingomyelinase activation associated factor (NSMAF), caspase 3 (Casp3), and interleukin (IL)-8, in patients with rotator cuff tendinopathy that had undergone surgical treatment. We included 44 participants that underwent arthroscopy, due to rotator cuff tendinopathy. Samples from the injured tendon were collected during arthroscopy, and RT-PCR was performed to determine gene expression. Pearson correlation analyses or U-Mann-Whitney test were performed to identify associations with the following parameters: sex, age at admission, body mass index, the presence of night pain, previous treatment (nonsteroidal anti-inflammatory drugs and/or steroids), medical history of the shoulder injury, upper subluxation of the humeral head, and the number of tendons injured. RT-PCR showed that the selected pro-inflammatory factors involved in the TNF-α signalling pathway expression levels were expressed in the tendon tissues. However, the levels of expression varied from patient to patient. Variations were over 250-fold for TNF-α, about 130-fold for TNFR1, NSMAF, and Casp3, and 1000-fold for IL-8. We could not confirm that any of the clinical parameters investigated were associated with the level of gene expression in the TNF-α pathway and IL-8.


Assuntos
Lesões do Manguito Rotador/imunologia , Tendões/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Caspase 3/genética , Feminino , Humanos , Interleucina-8/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Lesões do Manguito Rotador/cirurgia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/genética
10.
Viruses ; 13(2)2021 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-33668405

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging infectious disease of swine with zoonotic potential. Phylogenetic analysis suggests that PDCoV originated recently from a host-switching event between birds and mammals. Little is known about how PDCoV interacts with its differing hosts. Human-derived cell lines are susceptible to PDCoV infection. Herein, we compare the gene expression profiles of an established host swine cells to potential emerging host human cells after infection with PDCoV. Cell lines derived from intestinal lineages were used to reproduce the primary sites of viral infection in the host. Porcine intestinal epithelial cells (IPEC-J2) and human intestinal epithelial cells (HIEC) were infected with PDCoV. RNA-sequencing was performed on total RNA extracted from infected cells. Human cells exhibited a more pronounced response to PDCoV infection in comparison to porcine cells with more differentially expressed genes (DEGs) in human, 7486, in comparison to pig cells, 1134. On the transcriptional level, the adoptive host human cells exhibited more DEGs in response to PDCoV infection in comparison to the primary pig host cells, where different types of cytokines can control PDCoV replication and virus production. Key immune-associated DEGs and signaling pathways are shared between human and pig cells during PDCoV infection. These included genes related to the NF-kappa-B transcription factor family, the interferon (IFN) family, the protein-kinase family, and signaling pathways such as the apoptosis signaling pathway, JAK-STAT signaling pathway, inflammation/cytokine-cytokine receptor signaling pathway. MAP4K4 was unique in up-regulated DEGs in humans in the apoptosis signaling pathway. While similarities exist between human and pig cells in many pathways, our research suggests that the adaptation of PDCoV to the porcine host required the ability to down-regulate many response pathways including the interferon pathway. Our findings provide an important foundation that contributes to an understanding of the mechanisms of PDCoV infection across different hosts. To our knowledge, this is the first report of transcriptome analysis of human cells infected by PDCoV.


Assuntos
Infecções por Coronavirus/metabolismo , Células Epiteliais/virologia , Doenças dos Suínos/metabolismo , Transcriptoma , Animais , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Suínos
11.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540711

RESUMO

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation.


Assuntos
Polpa Dentária/patologia , Periodontite Periapical/patologia , Tecido Periapical/patologia , Pulpite/patologia , Animais , Antígenos de Neoplasias/imunologia , Carcinogênese/imunologia , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/fisiopatologia , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Cárie Dentária/fisiopatologia , Polpa Dentária/microbiologia , Dentina/irrigação sanguínea , Dentina/inervação , Dentina/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neoplasias Bucais/etiologia , Neoplasias Bucais/imunologia , Neoplasias Bucais/fisiopatologia , Rede Nervosa/fisiologia , Neuropeptídeos/metabolismo , Óxido Nítrico/fisiologia , Odontoblastos/fisiologia , Granuloma Periapical/etiologia , Granuloma Periapical/patologia , Tecido Periapical/microbiologia , Cisto Radicular/etiologia , Cisto Radicular/fisiopatologia
12.
Cancer Sci ; 112(4): 1383-1389, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33583097

RESUMO

Revertant (somatic) mosaicism is a spontaneous correction of a causative mutation in patients with congenital diseases. A relatively frequent event, revertant mosaicism may bring favorable outcomes that ameliorate disorders, and is therefore called "natural gene therapy." However, it has been revealed recently that "overcorrection" of inherited bone marrow failure in patients with sterile alpha motif domain containing 9 (SAMD9)/9L syndromes by revertant mosaicism induces myelodysplastic syndrome (MDS) with monosomy 7 that occasionally proceeds to acute myelogenous leukemia (AML). In this review, we interpret very complex mechanisms underlying MDS/AML in patients with SAMD9/9L syndromes. This includes multiple myeloid tumor suppressors on the long arm of chromosome 7, all of which act in a haploinsufficient fashion, and a difference in sensitivity to interferon between cells carrying a mutation and revertants. Overcorrection of mutants by somatic mosaicism is likely a novel mechanism in carcinogenesis.


Assuntos
Carcinogênese/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 7/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mosaicismo , Síndromes Mielodisplásicas/genética , Proteínas Supressoras de Tumor/genética
13.
Mol Cell Biol ; 41(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33526449

RESUMO

SHOC2 is a prototypical leucine-rich repeat protein that promotes downstream receptor tyrosine kinase (RTK)/RAS signaling and plays important roles in several cellular and developmental processes. Gain-of-function germ line mutations of SHOC2 drive the RASopathy Noonan-like syndrome, and SHOC2 mediates adaptive resistance to mitogen-activated protein kinase (MAPK) inhibitors. Similar to many scaffolding proteins, SHOC2 facilitates signal transduction by enabling proximal protein interactions and regulating the subcellular localization of its binding partners. Here, we review the structural features of SHOC2 that mediate its known functions, discuss these elements in the context of various binding partners and signaling pathways, and highlight areas of SHOC2 biology where a consensus view has not yet emerged.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Animais , Humanos , Sistema de Sinalização das MAP Quinases , Transdução de Sinais/fisiologia
14.
Nat Commun ; 12(1): 1158, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627648

RESUMO

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1-/- microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.


Assuntos
Colesterol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microglia/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/metabolismo , Doença de Niemann-Pick Tipo C/genética , Fagocitose/genética , Fagocitose/fisiologia , Proteômica/métodos
15.
Mol Cell ; 81(6): 1246-1259.e8, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33548203

RESUMO

The Integrator is a specialized 3' end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator's catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3' end-processing machineries points to distinct features and a unique architecture of the Integrator's catalytic module.


Assuntos
Complexos Multiproteicos , Terminação da Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
16.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(1): 43-48, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33565399

RESUMO

OBJECTIVE: To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis. METHODS: The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 µg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1ß and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. RESULTS: The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1ß (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1ß (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/ß-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/ß-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/ß-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. CONCLUSIONS: iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Animais , Exossomos/metabolismo , Humanos , Inflamassomos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato , Pirólise , Piroptose , Ratos
17.
Nat Commun ; 12(1): 1055, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594058

RESUMO

mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Arginina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Cinética , Lisina/metabolismo , Camundongos Nus , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação/efeitos dos fármacos
18.
Nat Commun ; 12(1): 121, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402676

RESUMO

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.


Assuntos
Coenzimas/química , Ditiocarb/análogos & derivados , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares/química , Compostos Organometálicos/química , Ubiquitina/química , Proteína com Valosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/genética , Coenzimas/metabolismo , Microscopia Crioeletrônica , Dissulfiram/química , Dissulfiram/metabolismo , Ditiocarb/química , Ditiocarb/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos Organometálicos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Dedos de Zinco
19.
Nat Commun ; 12(1): 241, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431824

RESUMO

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Crise Blástica/patologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/antagonistas & inibidores
20.
Methods Mol Biol ; 2260: 49-82, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405031

RESUMO

The epithelial cell is usually the first host cell that interacts with the microbiota present at mucosal surfaces. Although initially thought of as "bystander" cells with barrier function, the epithelial cell is now known to be a sentinel cell in the recognition and discrimination of commensal and pathogenic microorganisms and a key cell in initiating subsequent innate and adaptive immune responses. Here, we describe the main assays utilized in analyzing the activation of epithelial cell signaling (western blotting), transcription factors (TransAm), gene expression (quantitative reverse transcription PCR (qRT-PCR)), cytokine responses (ELISA, Luminex), and damage induction (lactate dehydrogenase (LDH) release). While our laboratory focuses on the epithelial response to Candida pathogens, these assays can be applied universally to analyze the activation of epithelial cells in response to any microbial pathogen.


Assuntos
Western Blotting , Candida/patogenicidade , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Animais , Sobrevivência Celular , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , L-Lactato Desidrogenase/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
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