RESUMO
Background: Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses. Methods: We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice (Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer (Cdx2-Cre +/Apc F/+), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells. Results: IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function. Conclusion: IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC.
Assuntos
Interleucina-17 , Camundongos Knockout , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Interleucina-17/metabolismo , Camundongos , Humanos , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Modelos Animais de DoençasRESUMO
Interleukin (IL)-17A and then IL-17F have been discovered through their roles in chronic inflammatory diseases. These cytokines share 50% of sequence homology and bind to the same receptor made of the IL-17RA et IL-17RC chains. While they have rather similar pro-inflammatory effects, slight differences exist depending on the cell type considered or whether there is TNF or not. Indeed, there is a synergistic effect of TNF and IL-17A or IL-17F on many cell types. In addition, the interactions between immune and stromal cells also modulate their effects which vary according to stromal cell subtype. The identification of IL-17A and IL-17F roles in inflammatory diseases, as psoriasis, has led to the development of inhibitors of those cytokines. Anti-IL-17A, then anti-IL-17A/F and now anti-IL-17RA have been approved for different diseases and are particularly efficient in psoriasis. Their use is expending to other diseases like psoriatic arthritis and spondyloarthritis. Last, the recent understanding of the importance of stromal cells during chronic inflammation explains the relative inefficacy of such inhibitors in some other diseases.
Title: IL-17A et IL-17F : de la découverte au ciblage thérapeutique - Un exemple de médecine translationnelle. Abstract: L'interleukine (IL)-17A puis l'IL-17F ont été découvertes tour à tour pour leur rôle joué dans les maladies inflammatoires chroniques. Elles ont une homologie de séquence d'environ 50 % et partagent le même récepteur formé des chaînes IL-17RA et IL-17RC. Si elles ont des effets pro-inflammatoires assez similaires, il existe néanmoins quelques différences selon le type cellulaire considéré et selon la présence ou non de TNF, autre cytokine avec laquelle elles ont une synergie d'action. La troisième variable venant moduler leurs effets réside dans les interactions entre cellules immunes et cellules stromales, qui, là encore, varient selon le type de cellules stromales. La mise en évidence de leur rôle dans le psoriasis a notamment conduit au développement d'inhibiteurs de l'IL-17A, puis à la fois de l'IL-17A et de l'IL-17F et enfin d'un de leurs récepteurs. Ces inhibiteurs sont utilisés avec succès dans cette pathologie, et leur indication a été étendue progressivement au rhumatisme psoriasique et à certaines formes de spondylarthrite. Enfin, la récente compréhension de l'importance des cellules stromales dans la réaction inflammatoire chronique permet d'expliquer l'efficacité variable de ces biothérapies dans certaines pathologies.
Assuntos
Produtos Biológicos , Interleucina-17 , Psoríase , Pesquisa Translacional Biomédica , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/fisiologia , Psoríase/tratamento farmacológico , Psoríase/imunologia , Animais , Produtos Biológicos/uso terapêutico , Produtos Biológicos/farmacologia , Inflamação/tratamento farmacológico , Descoberta de Drogas/tendências , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Receptores de Interleucina-17/fisiologia , Receptores de Interleucina-17/antagonistas & inibidoresRESUMO
Oropharyngeal candidiasis (OPC) is the most common human fungal infection, arising typically from T cell immune impairments. IL-17 and IL-22 contribute individually to OPC responses, but here we demonstrate that the combined actions of both cytokines are essential for resistance to OPC. Mice lacking IL-17RA and IL-22RA1 exhibited high fungal loads in esophagus- and intestinal tract, severe weight loss, and symptoms of colitis. Ultimately, mice succumbed to infection. Dual loss of IL-17RA and IL-22RA impaired expression of small proline rich proteins (SPRRs), a class of antimicrobial effectors not previously linked to fungal immunity. Sprr2a1 exhibited direct candidacidal activity in vitro, and Sprr1-3a-/- mice were susceptible to OPC. Thus, cooperative actions of Type 17 cytokines mediate oral mucosal anti-Candida defenses and reveal a role for SPRRs.
Assuntos
Candidíase Bucal , Interleucina-17 , Interleucina 22 , Interleucinas , Camundongos Knockout , Animais , Camundongos , Candida albicans/imunologia , Candidíase Bucal/imunologia , Candidíase Bucal/microbiologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/imunologia , Receptores de Interleucina-17/metabolismoRESUMO
INTRODUCTION: The treatment of multiple myeloma (MM) is evolving rapidly. Quadruplet regimens incorporating proteasome inhibitors, immunomodulatory drugs (IMiDs), and CD38 monoclonal antibodies have emerged as standard-of-care options for newly diagnosed MM, and numerous novel therapies have been approved for relapsed/refractory MM. However, there remains a need for novel options in multiple settings, including refractoriness to frontline standards of care. AREAS COVERED: Targeting degradation of IKZF1 and IKZF3 - Ikaros and Aiolos - through modulation of cereblon, an E3 ligase substrate recruiter/receptor, is a key mechanism of action of the IMiDs and the CELMoD agents. Two CELMoD agents, iberdomide and mezigdomide, have demonstrated substantial preclinical and clinical activity in MM and have entered phase 3 investigation. Using a literature search methodology comprising searches of PubMed (unlimited time-frame) and international hematology/oncology conference abstracts (2019-2023), this paper reviews the importance of Ikaros and Aiolos in MM, the mechanism of action of the IMiDs and CELMoD agents and their relative potency for targeting Ikaros and Aiolos, and preclinical and clinical data on iberdomide and mezigdomide. EXPERT OPINION: Emerging data suggest that iberdomide and mezigdomide have promising activity, including in IMiD-resistant settings and, pending phase 3 findings, may provide additional treatment options for patients with MM.
Assuntos
Fator de Transcrição Ikaros , Mieloma Múltiplo , Talidomida , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Fator de Transcrição Ikaros/metabolismo , Talidomida/uso terapêutico , Talidomida/análogos & derivados , Talidomida/farmacologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Proteólise/efeitos dos fármacos , Terapia de Alvo Molecular , Agentes de Imunomodulação/uso terapêutico , Agentes de Imunomodulação/farmacologia , Ensaios Clínicos como Assunto , Animais , Piperidonas , Morfolinas , Receptores de Interleucina-17 , Proteínas Adaptadoras de Transdução de Sinal , FtalimidasRESUMO
Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Transcrição Gênica , Ubiquitinação , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Culina/metabolismo , Proteínas Culina/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Microscopia Crioeletrônica , Células HEK293 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Dano ao DNA , RNA Polimerase II/metabolismo , Ligação Proteica , Reparo por Excisão , Proteínas de Transporte , DNA Helicases , Fatores de Transcrição , Receptores de Interleucina-17RESUMO
BACKGROUND: We previously identified Il17RB, a member of the IL17 superfamily, as a candidate marker gene for endometrial aging. While IL17RB has been linked to inflammation and malignancies in several organ systems, its function in the endometrium has not been investigated and is thus poorly understood. In the present study, we performed a functional analysis of this receptor with the aim of determining the effects of its age-associated overexpression on the uterine environment. METHODS: We analyzed IL17RB-related signaling pathways and downstream gene expression in an immortalized human endometrial glandular epithelial cell line ("hEM") forced to express the receptor via lentiviral transduction ("IL17RB-hEM"). We also prepared endometrial organoids from human endometrial tissue sourced from hysterectomy patients ("patient-derived EOs") and exposed them to cytokines that are upregulated by IL17RB expression to investigate changes in organoid-forming capacity and senescence markers. We analyzed RNA-seq data (GEO accession number GSE132886) from our previous study to identify the signaling pathways associated with altered IL17RB expression. We also analyzed the effects of the JNK pathway on organoid-forming capacity. RESULTS: Stimulation with interleukin 17B enhanced the NF-κB pathway in IL17RB-hEM, resulting in significantly elevated expression of the genes encoding the senescence associated secretory phenotype (SASP) factors IL6, IL8, and IL1ß. Of these cytokines, IL1ß inhibited endometrial organoid growth. Bioinformatics analysis showed that the JNK signaling pathway was associated with age-related variation in IL17RB expression. When IL17RB-positive cells were cultured in the presence of IL17B, their organoid-forming capacity was slightly but non-significantly lower than in unexposed IL17RB-positive cells, but when IL17B was paired with a JNK inhibitor (SP600125), it was restored to control levels. Further, IL1ß exposure significantly reduced organoid-forming capacity and increased p21 expression in endometrial organoids relative to non-exposure (control), but when IL1ß was paired with SP600125, both indicators were restored to levels comparable to the control condition. CONCLUSIONS: We have revealed an association between IL17RB, whose expression increases in the endometrial glandular epithelium with advancing age, and cellular senescence. Using human endometrial organoids as in vitro model, we found that IL1ß inhibits cell proliferation and leads to endometrial senescence via the JNK pathway.
Assuntos
Senescência Celular , Endométrio , Receptores de Interleucina-17 , Transdução de Sinais , Humanos , Feminino , Endométrio/metabolismo , Endométrio/citologia , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genética , Senescência Celular/genética , Organoides/metabolismo , Linhagem CelularRESUMO
Acamprosate is a Food and Drug Administration (FDA) approved medication for the treatment of alcohol use disorder (AUD). However, only a subset of patients achieves optimal treatment outcomes. Currently, no biological measures are utilized to predict response to acamprosate treatment. We applied our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Specifically, our previous open-label acamprosate clinical trial recruited 442 patients with AUD who were treated with acamprosate for three months. We first performed proteomics using baseline plasma samples to identify potential biomarkers associated with acamprosate treatment outcomes. Next, we applied our established "proteomics-informed genome-wide association study (GWAS)" research strategy, and identified 12 proteins, including interleukin-17 receptor B (IL17RB), associated with acamprosate treatment response.â A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variantgenotypes for all of those IL17RB SNPs were protective for alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. In summary, this study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying acamprosate treatment response phenotypes. (The ClinicalTrials.gov Identifier: NCT00662571).
Assuntos
Acamprosato , Dissuasores de Álcool , Alcoolismo , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Proteômica , Receptores de Interleucina-17 , Humanos , Acamprosato/uso terapêutico , Polimorfismo de Nucleotídeo Único/genética , Alcoolismo/genética , Alcoolismo/tratamento farmacológico , Masculino , Feminino , Proteômica/métodos , Dissuasores de Álcool/uso terapêutico , Pessoa de Meia-Idade , Adulto , Receptores de Interleucina-17/genética , Resultado do Tratamento , Genômica/métodos , Biomarcadores/sangue , Taurina/análogos & derivados , Taurina/uso terapêuticoRESUMO
The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.
Assuntos
Motivos de Aminoácidos , Microscopia Crioeletrônica , Proteínas Culina , Receptores de Interleucina-17 , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Culina/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Células HEK293 , Multimerização Proteica , Sequência Conservada , Ligação Proteica , Modelos MolecularesRESUMO
The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID.
Assuntos
Tubas Uterinas , Gonorreia , Inflamação , Interleucina-17 , Neisseria gonorrhoeae , Adulto , Feminino , Humanos , Citocinas/metabolismo , Epitélio/patologia , Epitélio/microbiologia , Tubas Uterinas/microbiologia , Tubas Uterinas/patologia , Tubas Uterinas/imunologia , Gonorreia/imunologia , Gonorreia/microbiologia , Gonorreia/patologia , Inflamação/patologia , Inflamação/microbiologia , Interleucina-17/metabolismo , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/patogenicidade , Doença Inflamatória Pélvica/microbiologia , Doença Inflamatória Pélvica/patologia , Doença Inflamatória Pélvica/imunologia , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genéticaRESUMO
Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.
Assuntos
Proteínas de Ciclo Celular , Oxindóis , Proteólise , Ubiquitina-Proteína Ligases , Humanos , Animais , Proteólise/efeitos dos fármacos , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Oxindóis/farmacologia , Oxindóis/metabolismo , Oxindóis/química , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Células HEK293 , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Azepinas/farmacologia , Azepinas/química , Azepinas/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Proteínas que Contêm Bromodomínio , Receptores de Interleucina-17RESUMO
Defective clearance of apoptotic cells due to impaired efferocytosis sustains error in self-tolerance that exacerbates rheumatoid arthritis (RA). However, the molecular determinant that directly or specifically impairs efferocytosis in RA is not yet studied. We identified a new perspective that IL-17A significantly impedes efferocytosis via preferential activation of the JAK/STAT-3/ADAM17 signaling axis. In contrast, disruption of the IL-17A/IL-17RA interaction using cyanidin or silencing of IL-17RA obstructed JAK/STAT-3 activation that further abolished ADAM17 expression. Subsequent depletion of ADAM17 inhibited the shedding of Mer tyrosine kinase receptor (MERTK), which significantly increased apoptotic cell intake and restored efferocytosis in adjuvant-induced arthritic (AA) model. Concomitantly, the amplification of the efferocytosis process due to IL-17A/IL-17RA interaction disruption was sensitive to mitochondrial fission mediated via Drp-1 phosphorylation downstream of STAT-3 inhibition. As expected, cyanidin treated AA synovial macrophages that exhibited increased efferocytosis demonstrated a phenotypic shift towards CD163 anti-inflammatory phenotype in a STAT-5 dependent manner. Similar results were obtained in IL-17A-sensitized AA synovial macrophages treated with S3I-201 (a STAT-3 inhibitor) indicating that IL-17A influences efferocytosis via the STAT-3 pathway. In view of our previous work where cyanidin restored Th17/Treg balance, our present investigation fulfils a critical gap by providing scientific validation that cyanidin escalated PD-L1 expression during the efferocytosis process that could have impacted the restoration of Th17/Treg balance in an AA model. Together, these data corroborate the hypothesis that IL-17A signaling can impair efferocytosis via regulating STAT-3/ADAM17/FL-MERTK axis and that its inhibition can amplify a pro-resolution signal against RA progression.
Assuntos
Artrite Reumatoide , Antígeno B7-H1 , Interleucina-17 , Macrófagos , Receptores de Interleucina-17 , Fator de Transcrição STAT3 , Transdução de Sinais , Interleucina-17/metabolismo , Animais , Fator de Transcrição STAT3/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/tratamento farmacológico , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Camundongos , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genética , Antocianinas/farmacologia , Artrite Experimental/imunologia , Humanos , Modelos Animais de Doenças , Fagocitose/efeitos dos fármacos , Masculino , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Camundongos Endogâmicos C57BL , Apoptose/efeitos dos fármacos , EferocitoseRESUMO
The IL-17 pathway displays remarkably diverse functional modes between different subphyla, classes, and even orders, yet its driving factors remains elusive. Here, we demonstrate that the IL-17 pathway originated through domain shuffling between a Toll-like receptor (TLR)/IL-1R pathway and a neurotrophin-RTK (receptor-tyrosine-kinase) pathway (a Trunk-Torso pathway). Unlike other new pathways that evolve independently, the IL-17 pathway remains intertwined with its donor pathways throughout later evolution. This intertwining not only influenced the gains and losses of domains and components in the pathway but also drove the diversification of the pathway's functional modes among animal lineages. For instance, we reveal that the crustacean female sex hormone, a neurotrophin inducing sex differentiation, could interact with IL-17Rs and thus be classified as true IL-17s. Additionally, the insect prothoracicotropic hormone, a neurotrophin initiating ecdysis in Drosophila by binding to Torso, could bind to IL-17Rs in other insects. Furthermore, IL-17R and TLR/IL-1R pathways maintain crosstalk in amphioxus and zebrafish. Moreover, the loss of the Death domain in the pathway adaptor connection to IκB kinase and stress-activated protein kinase (CIKSs) dramatically reduced their abilities to activate nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) in amphioxus and zebrafish. Reinstating this Death domain not only enhanced NF-κB/AP-1 activation but also strengthened anti-bacterial immunity in zebrafish larvae. This could explain why the mammalian IL-17 pathway, whose CIKS also lacks Death, is considered a weak signaling activator, relying on synergies with other pathways. Our findings provide insights into the functional diversity of the IL-17 pathway and unveil evolutionary principles that could govern the pathway and be used to redesign and manipulate it.
Assuntos
Interleucina-17 , Transdução de Sinais , Receptores Toll-Like , Animais , Interleucina-17/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genética , Evolução Molecular , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genéticaRESUMO
Fracture healing is a complex series of events that requires a local inflammatory reaction to initiate the reparative process. This inflammatory reaction is important for stimulating the migration and proliferation of mesenchymal progenitor cells from the periosteum and surrounding tissues to form the cartilaginous and bony calluses. The proinflammatory cytokine interleukin (IL)-17 family has gained attention for its potential regenerative effects; however, the requirement of IL-17 signaling within mesenchymal progenitor cells for normal secondary fracture healing remains unknown. The conditional knockout of IL-17 receptor a (Il17ra) in mesenchymal progenitor cells was achieved by crossing Il17raF/F mice with Prx1-cre mice to generate Prx1-cre; Il17raF/F mice. At 3 months of age, mice underwent experimental unilateral mid-diaphyseal femoral fractures and healing was assessed by micro-computed tomography (µCT) and histomorphometric analyses. The effects of IL-17RA signaling on the osteogenic differentiation of fracture-activated periosteal cells was investigated in vitro. Examination of the intact skeleton revealed that the conditional knockout of Il17ra decreased the femoral cortical porosity but did not affect any femoral trabecular microarchitectural indices. After unilateral femoral fractures, Il17ra conditional knockout impacted the cartilage and bone composition of the fracture callus that was most evident early in the healing process (day 7 and 14 post-fracture). Furthermore, the in vitro treatment of fracture-activated periosteal cells with IL-17A inhibited osteogenesis. This study suggests that IL-17RA signaling within Prx1+ mesenchymal progenitor cells can influence the early stages of endochondral ossification during fracture healing.
Assuntos
Fraturas do Fêmur , Proteínas de Homeodomínio , Células-Tronco Mesenquimais , Receptores de Interleucina-17 , Animais , Camundongos , Consolidação da Fratura , Inflamação , Osteogênese , Microtomografia por Raio-X , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Proteínas de Homeodomínio/metabolismoRESUMO
BACKGROUND: Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with mild cognitive impairment and motor incoordination. Rats with chronic hyperammonemia reproduce these alterations. Motor incoordination in hyperammonemic rats is due to increased GABAergic neurotransmission in cerebellum, induced by neuroinflammation, which enhances TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway activation. The initial events by which hyperammonemia triggers activation of this pathway remain unclear. MHE in cirrhotic patients is triggered by a shift in inflammation with increased IL-17. The aims of this work were: (1) assess if hyperammonemia increases IL-17 content and membrane expression of its receptor in cerebellum of hyperammonemic rats; (2) identify the cell types in which IL-17 receptor is expressed and IL-17 increases in hyperammonemia; (3) assess if blocking IL-17 signaling with anti-IL-17 ex-vivo reverses activation of glia and of the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. RESULTS: IL-17 levels and membrane expression of the IL-17 receptor are increased in cerebellum of rats with hyperammonemia and MHE, leading to increased activation of IL-17 receptor in microglia, which triggers activation of STAT3 and NF-kB, increasing IL-17 and TNFα levels, respectively. TNFα released from microglia activates TNFR1 in Purkinje neurons, leading to activation of NF-kB and increased IL-17 and TNFα also in these cells. Enhanced TNFR1 activation also enhances activation of the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway which mediates microglia and astrocytes activation. CONCLUSIONS: All these steps are triggered by enhanced activation of IL-17 receptor in microglia and are prevented by ex-vivo treatment with anti-IL-17. IL-17 and IL-17 receptor in microglia would be therapeutic targets to treat neurological impairment in patients with MHE.
Assuntos
Cerebelo , Hiperamonemia , Microglia , Ratos Wistar , Receptores de Interleucina-17 , Animais , Hiperamonemia/metabolismo , Microglia/metabolismo , Cerebelo/metabolismo , Masculino , Ratos , Receptores de Interleucina-17/metabolismo , Doenças Neuroinflamatórias/metabolismo , Interleucina-17/metabolismo , Encefalopatia Hepática/metabolismo , Transdução de Sinais , Modelos Animais de DoençasRESUMO
Ulcerative colitis (UC) is a complicated and recurrent intestinal disease. Currently available drugs for UC treatment are scarce, therefore, novel therapeutic drugs for the UC are urgently to be developed. Gingerenone A (GA) is a phenolic compound known for its anti-inflammatory effect, but its effect on UC remains unknown. Here, it is shown that GA protects mice against UC, which is closely associated with inhibiting intestinal mucosal inflammation and enhancing intestinal barrier integrity in vivo and in vitro. Of note, RNA sequencing analysis demonstrates an evident correlation with IL-17 signaling pathway after GA treatment, and this effect is further corroborated by Western blot. Mechanistically, GA directly interacts with IL-17RA protein through pull-down, surface plasmon resonance analysis and molecular dynamics simulation. Importantly, lentivirus-mediated IL-17RA/Act1 knock-down or GA co-treatment with brodalumab/ixekizumab significantly impairs the protective effects of GA against DSS-induced inflammation and barrier dysfunction, suggesting a critical role of IL-17RA signaling for GA-mediated protection against UC. Overall, these results indicate that GA is an effective agent against UC mainly through the direct binding of IL-17RA to inhibit inflammatory signaling activation.
Assuntos
Colite Ulcerativa , Modelos Animais de Doenças , Mucosa Intestinal , Receptores de Interleucina-17 , Animais , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Função da Barreira Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Covalent DNA-protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA-protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.
Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Adutos de DNA/metabolismo , Adutos de DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo por Excisão , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição , Transcrição Gênica , Ubiquitinação , Raios UltravioletaAssuntos
Dermatite Atópica , Modelos Animais de Doenças , Progressão da Doença , Receptores de Interleucina-17 , Dermatite Atópica/imunologia , Dermatite Atópica/tratamento farmacológico , Animais , Camundongos , Receptores de Interleucina-17/imunologia , Receptores de Interleucina-17/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia , HumanosRESUMO
The E3 ligase-degron interaction determines the specificity of the ubiquitinâproteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.
Assuntos
Microscopia Crioeletrônica , Proteína NEDD8 , Receptores de Interleucina-17 , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/química , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Prolina/metabolismo , Multimerização Proteica , Células HEK293 , Ligação Proteica , Especificidade por Substrato , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas Culina/metabolismo , Proteínas Culina/química , Proteínas Culina/genética , DegronsRESUMO
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Assuntos
DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , Proteólise , RNA Polimerase II , Transcrição Gênica , Humanos , Proteínas de Transporte , DNA/metabolismo , DNA/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Interleucina-17 , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Helicobacter pylori is a Gram-negative pathogen that colonizes the stomach, induces inflammation, and drives pathological changes in the stomach tissue, including gastric cancer. As the principal cytokine produced by Th17 cells, IL-17 mediates protective immunity against pathogens by inducing the activation and mobilization of neutrophils. Whereas IL-17A is largely produced by lymphocytes, the IL-17 receptor is expressed in epithelial cells, fibroblasts, and hematopoietic cells. Loss of the IL-17RA in mice results in impaired antimicrobial responses to extracellular bacteria. In the context of H. pylori infection, this is compounded by extensive inflammation in Il17ra-/- mice. In this study, Foxa3creIl17rafl/fl (Il17raΔGI-Epi) and Il17rafl/fl (control) mice were used to test the hypothesis that IL-17RA signaling, specifically in epithelial cells, protects against severe inflammation after H. pylori infection. The data indicate that Il17raΔGI-Epi mice develop increased inflammation compared with controls. Despite reduced Pigr expression, levels of IgA increased in the gastric wash, suggesting significant increase in Ag-specific activation of the T follicular helper/B cell axis. Gene expression analysis of stomach tissues indicate that both acute and chronic responses are significantly increased in Il17raΔGI-Epi mice compared with controls. These data suggest that a deficiency of IL-17RA in epithelial cells is sufficient to drive chronic inflammation and hyperactivation of the Th17/T follicular helper/B cell axis but is not required for recruitment of polymorphonuclear neutrophils. Furthermore, the data suggest that fibroblasts can produce chemokines in response to IL-17 and may contribute to H. pylori-induced inflammation through this pathway.