RESUMEN
Targeting tumor-infiltrating regulatory T (TI-Treg) cells is a potential strategy for cancer therapy. The ATPase p97 in complex with cofactors (such as Npl4) has been investigated as an antitumor drug target; however, it is unclear whether p97 has a function in immune cells or immunotherapy. Here we show that thonzonium bromide is an inhibitor of the interaction of p97 and Npl4 and that this p97-Npl4 complex has a critical function in TI-Treg cells. Thonzonium bromide boosts antitumor immunity without affecting peripheral Treg cell homeostasis. The p97-Npl4 complex bridges Stat3 with E3 ligases PDLIM2 and PDLIM5, thereby promoting Stat3 degradation and enabling TI-Treg cell development. Collectively, this work shows an important role for the p97-Npl4 complex in controlling Treg-TH17 cell balance in tumors and identifies possible targets for immunotherapy.
RESUMEN
Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.
Asunto(s)
ARN Circular/metabolismo , ARN Circular/fisiología , eIF-2 Quinasa/metabolismo , Adolescente , Adulto , Enfermedades Autoinmunes/genética , Línea Celular , Endorribonucleasas/metabolismo , Femenino , Humanos , Inmunidad Innata/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/genética , Persona de Mediana Edad , Fosforilación , ARN/metabolismo , Empalme del ARN/genética , Estabilidad del ARN/fisiología , ARN Circular/genética , ARN Bicatenario/metabolismo , Virosis/metabolismo , eIF-2 Quinasa/inmunologíaRESUMEN
Hepatocellular carcinoma (HCC) formation is a multi-step pathological process that involves evolution of a heterogeneous immunosuppressive tumor microenvironment. However, the specific cell populations involved and their origins and contribution to HCC development remain largely unknown. Here, comprehensive single-cell transcriptome sequencing was applied to profile rat models of toxin-induced liver tumorigenesis and HCC patients. Specifically, we identified three populations of hepatic parenchymal cells emerging during HCC progression, termed metabolic hepatocytes (HCMeta ), Epcam+ population with differentiation potential (EP+Diff ) and immunosuppressive malignant transformation subset (MTImmu ). These distinct subpopulations form an oncogenic trajectory depicting a dynamic landscape of hepatocarcinogenesis, with signature genes reflecting the transition from EP+Diff to MTImmu . Importantly, GPNMB+ Gal-3+ MTImmu cells exhibit both malignant and immunosuppressive properties. Moreover, SOX18 is required for the generation and malignant transformation of GPNMB+ Gal-3+ MTImmu cells. Enrichment of the GPNMB+ Gal-3+ MTImmu subset was found to be associated with poor prognosis and a higher rate of recurrence in patients. Collectively, we unraveled the single-cell HCC progression atlas and uncovered GPNMB+ Gal-3+ parenchymal cells as a major subset contributing to the immunosuppressive microenvironment thus malignance in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animales , Ratas , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hepatocitos , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Terapia de Inmunosupresión , Microambiente Tumoral , Factores de Transcripción SOXF , Glicoproteínas de Membrana/genéticaRESUMEN
Immune responses need to be tightly controlled to avoid excessive inflammation and prevent unwanted host damage. Here we report that germinal center kinase MST4 responded dynamically to bacterial infection and acted as a negative regulator of inflammation. We found that MST4 directly interacted with and phosphorylated the adaptor TRAF6 to prevent its oligomerization and autoubiquitination. Accordingly, MST4 did not inhibit lipopolysaccharide-induced cytokine production in Traf6(-/-) embryonic fibroblasts transfected to express a mutant form of TRAF6 that cannot be phosphorylated at positions 463 and 486 (with substitution of alanine for threonine at those positions). Upon developing septic shock, mice in which MST4 was knocked down showed exacerbated inflammation and reduced survival, whereas heterozygous deletion of Traf6 (Traf6(+/-)) alleviated such deleterious effects. Our findings reveal a mechanism by which TRAF6 is regulated and highlight a role for MST4 in limiting inflammatory responses.
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Inflamación/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Masculino , Ratones , Persona de Mediana Edad , Sepsis/sangre , Choque Séptico/inducido químicamente , Choque Séptico/metabolismoRESUMEN
The Hippo signaling pathway plays an essential role in organ size control and tumorigenesis. Loss of Hippo signal and hyper-activation of the downstream oncogenic YAP signaling are commonly observed in various types of cancers. We previously identified STRN3-containing PP2A phosphatase as a negative regulator of MST1/2 kinases (i.e., Hippo) in gastric cancer (GC), opening the possibility of selectively targeting the PP2Aa-STRN3-MST1/2 axis to recover Hippo signaling against cancer. Here, we further discovered 1) disulfiram (DSF), an FDA-approved drug, which can similarly block the binding of STRN3 to PP2A core enzyme and 2) CX-6258 (CX), a chemical inhibitor, that can disrupt the interaction between STRN3 and MST1/2, both allowing reactivation of Hippo activity to inhibit GC. More importantly, we found these two compounds, via an MST1/2 kinase-dependent manner, inhibit DNA repair to sensitize GC towards chemotherapy. In addition, we identified thiram, a structural analog of DSF, can function similarly to inhibit cancer cell proliferation or enhance chemotherapy sensitivity. Interestingly, inclusion of copper ion enhanced such effects of DSF and thiram on GC treatment. Overall, this work demonstrated that pharmacological targeting of the PP2Aa-STRN3-MST1/2 axis by drug compounds can potently recover Hippo signal for tumor treatment.
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Disulfiram , Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Neoplasias Gástricas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Disulfiram/farmacología , Línea Celular Tumoral , Animales , Antineoplásicos/farmacología , Transducción de Señal/efectos de los fármacos , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento de Hepatocito/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genéticaRESUMEN
The Hippo-YAP signaling pathway plays an essential role in epithelial cells during intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here, we report a stroma-epithelium ISLR-YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can inhibit Hippo signaling and activate YAP in epithelial cells. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cell-specific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings provide new insights into the signaling crosstalk between stroma and epithelium during tissue regeneration and tumorigenesis.
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Neoplasias Colorrectales/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Animales , Neoplasias Colorrectales/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Células HCT116 , Células HEK293 , Células HT29 , Vía de Señalización Hippo , Humanos , Enfermedades Inflamatorias del Intestino/genética , Mucosa Intestinal/metabolismo , Masculino , Ratones , Mutación , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.
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Fibroblastos Asociados al Cáncer , Neoplasias Colorrectales , Humanos , Fibroblastos Asociados al Cáncer/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular Tumoral , Transducción de Señal , Carcinogénesis/patología , Neoplasias Colorrectales/patología , Proliferación Celular , Microambiente Tumoral , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/farmacología , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/farmacologíaRESUMEN
Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HIT-type containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.
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Proteínas Portadoras , Cromatina , Pulmón , Animales , Ratones , Proteína Morfogenética Ósea 4/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Pulmón/metabolismo , Morfogénesis/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND & AIMS: Remodeling the tumor microenvironment is a critical strategy for treating advanced hepatocellular carcinoma (HCC). Yet, how distinct cell populations in the microenvironment mediate tumor resistance to immunotherapies, such as anti-PD-1, remains poorly understood. METHODS: We analyzed the transcriptomic profile, at a single-cell resolution, of tumor tissues from patients with HCC scheduled to receive anti-PD-1-based immunotherapy. Our comparative analysis and experimental validation using flow cytometry and histopathological analysis uncovered a discrete subpopulation of cells associated with resistance to anti-PD-1 treatment in patients and a rat model. A TurboID-based proximity labeling approach was deployed to gain mechanistic insights into the reprogramming of the HCC microenvironment. RESULTS: We identified CD10+ALPL+ neutrophils as being associated with resistance to anti-PD-1 treatment. These neutrophils exhibited a strong immunosuppressive activity by inducing an apparent "irreversible" exhaustion of T cells in terms of cell number, frequency, and gene profile. Mechanistically, CD10+ALPL+ neutrophils were induced by tumor cells, i.e., tumor-secreted NAMPT reprogrammed CD10+ALPL+ neutrophils through NTRK1, maintaining them in an immature state and inhibiting their maturation and activation. CONCLUSIONS: Collectively, our results reveal a fundamental mechanism by which CD10+ALPL+ neutrophils contribute to tumor immune escape from durable anti-PD-1 treatment. These data also provide further insights into novel immunotherapy targets and possible synergistic treatment regimens. IMPACT AND IMPLICATIONS: Herein, we discovered that tumor cells reprogrammed CD10+ALPL+ neutrophils to induce the "irreversible" exhaustion of T cells and hence allow tumors to escape from the intended effects of anti-PD-1 treatment. Our data provided a new theoretical basis for the elucidation of special cell populations and revealed a molecular mechanism underpinning resistance to immunotherapy. Targeting these cells alongside existing immunotherapy could be looked at as a potentially more effective therapeutic approach.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Ratas , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Linfocitos T , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neutrófilos , Inmunoterapia/métodos , Microambiente Tumoral , Linfocitos T CD8-positivos , Fosfatasa AlcalinaRESUMEN
Gastric cancer (GC) is an aggressive malignant disease which still lacks effective early diagnosis markers and targeted therapies, representing the fourth-leading cause of cancer-associated death worldwide. The Hippo signaling pathway plays crucial roles in organ size control and tissue homeostasis under physiological conditions, yet its aberrations have been closely associated with several hallmarks of cancer. The last decade witnessed a burst of investigations dissecting how Hippo dysregulation contributes to tumorigenesis, highlighting the therapeutic potential of targeting this pathway for tumor intervention. In this review, we systemically document studies on the Hippo pathway in the contexts of gastric tumor initiation, progression, metastasis, acquired drug resistance, and the emerging development of Hippo-targeting strategies. By summarizing major open questions in this field, we aim to inspire further in-depth understanding of Hippo signaling in GC development, as well as the translational implications of targeting Hippo for GC treatment.
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Vía de Señalización Hippo , Neoplasias Gástricas , Humanos , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Señalizadoras YAP , Factores de Transcripción/metabolismo , Transformación Celular NeoplásicaRESUMEN
The Yes-associated protein (YAP) is a major oncoprotein responsible for cell proliferation control. YAP's oncogenic activity is regulated by both the Hippo kinase cascade and uniquely by a mechanical-force-induced actin remodeling process. Inspired by reports that ovarian cancer cells specifically accumulate the phosphatase protein ALPP on lipid rafts that physically link to actin cytoskeleton, we developed a molecular self-assembly (MSA) technology that selectively halts cancer cell proliferation by inactivating YAP. We designed a ruthenium-complex-peptide precursor molecule that, upon cleavage of phosphate groups, undergoes self-assembly to form nanostructures specifically on lipid rafts of ovarian cancer cells. The MSAs exert potent, cancer-cell-specific antiproliferative effects in multiple cancer cell lines and in mouse xenograft tumor models. Our work illustrates how basic biochemical insights can be exploited as the basis for a nanobiointerface fabrication technology which links nanoscale protein activities at specific subcellular locations to molecular biological activities to suppress cancer cell proliferation.
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Neoplasias Ováricas , Proteínas Serina-Treonina Quinasas , Actinas , Animales , Femenino , Humanos , Microdominios de Membrana , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export.
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Proteínas Nucleares/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN , Transporte de ARN/genética , ARN Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/genética , Exosomas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Unión Proteica , ARN Helicasas/genética , Estabilidad del ARN/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factores de Transcripción/genéticaRESUMEN
Toll-like receptors (TLRs) are key players of the innate immune system and contribute to inflammation and pathogen clearance. Although TLRs have been extensively studied, it remains unclear how exactly bacterial lipopolysaccharide (LPS)-induced conformational changes of the extracellular domain of the TLRs trigger the dimerization of their intracellular domain across the plasma membrane and thereby stimulate downstream signaling. Here, using LPS-stimulated THP-1-derived macrophages and murine macrophages along with immunoblotting and immunofluorescence and quantitative analyses, we report that in response to inflammatory stimuli, the coiled-coil protein TRAF3-interacting JNK-activating modulator (T3JAM) associates with TLR4, promotes its translocation to lipid rafts, and thereby enhances macrophage-mediated inflammation. T3JAM overexpression increased and T3JAM depletion decreased TLR4 signaling through both the MyD88-dependent pathway and TLR4 endocytosis. Importantly, deletion or mutation of T3JAM to disrupt its coiled-coil-mediated homoassociation abrogated TLR4 recruitment to lipid rafts. Consistently, T3JAM depletion in mice dampened TLR4 signaling and alleviated LPS-induced inflammatory damage. Collectively, our findings reveal an additional molecular mechanism by which TLR4 activity is regulated and suggest that T3JAM may function as a molecular clamp to "tighten up" TLR4 and facilitate its translocation to lipid rafts.
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Proteínas Portadoras/fisiología , Inflamación/patología , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas , Transducción de Señal , Receptor Toll-Like 4/genéticaRESUMEN
The mammalian STE20-like protein kinase 1 (MST1)-MOB kinase activator 1 (MOB1) complex has been shown to suppress the oncogenic activity of Yes-associated protein (YAP) in the mammalian Hippo pathway, which is involved in the development of multiple tumors, including pancreatic cancer (PC). However, it remains unclear whether other MST-MOB complexes are also involved in regulating Hippo-YAP signaling and have potential roles in PC. Here, we report that mammalian STE20-like kinase 4 (MST4), a distantly related ortholog of the MST1 kinase, forms a complex with MOB4 in a phosphorylation-dependent manner. We found that the overall structure of the MST4-MOB4 complex resembles that of the MST1-MOB1 complex, even though the two complexes exhibited opposite biological functions in PC. In contrast to the tumor-suppressor effect of the MST1-MOB1 complex, the MST4-MOB4 complex promoted growth and migration of PANC-1 cells. Moreover, expression levels of MST4 and MOB4 were elevated in PC and were positively correlated with each other, whereas MST1 expression was down-regulated. Because of divergent evolution of key interface residues, MST4 and MOB4 could disrupt assembly of the MST1-MOB1 complex through alternative pairing and thereby increased YAP activity. Collectively, these findings identify the MST4-MOB4 complex as a noncanonical regulator of the Hippo-YAP pathway with an oncogenic role in PC. Our findings highlight that although MST-MOB complexes display some structural conservation, they functionally diverged during their evolution.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Oncogenes , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Regulación hacia Abajo , Células HEK293 , Factor de Crecimiento de Hepatocito/química , Vía de Señalización Hippo , Humanos , Neoplasias Pancreáticas/patología , Fosforilación , Pronóstico , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Factores de Transcripción , Regulación hacia Arriba , Proteínas Señalizadoras YAPRESUMEN
RIG-I is a well-studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4-Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG-I. The p97 complex is able to directly bind both non-ubiquitinated RIG-I and the E3 ligase RNF125, promoting K48-linked ubiquitination of RIG-I at residue K181. Viral infection significantly strengthens the interaction between RIG-I and the p97 complex by a conformational change of RIG-I that exposes the CARDs and through K63-linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG-I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.
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Adenosina Trifosfatasas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Adenosina Trifosfatasas/genética , Animales , Línea Celular , Células HeLa , Humanos , Ratones , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Receptores de Ácido Retinoico/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/prevención & control , Replicación Viral/fisiologíaRESUMEN
The Hh pathway controls many morphogenetic processes in metazoans and plays important roles in numerous pathologies and in cancer. Hh signaling is mediated by the activity of the Gli/Ci family of transcription factors. Several studies in Drosophila have shown that ubiquitination by the ubiquitin E3 ligases Slimb and Rdx(Hib) plays a crucial role in controlling Ci stability dependent on the levels of Hh signals. If Hh levels are low, Slimb adds K11- and K48-linked poly-ubiquitin chains on Ci resulting in partial degradation. Ubiquitin E2 enzymes are pivotal in determining the topologies of ubiquitin chains. However, which E2 enzymes participate in the selective ubiquitination-degradation of Ci remains elusive. Here, we find that the E2 enzyme UbcD1 negatively regulates Hh signaling activity in Drosophila wing disks. Genetic and biochemical analyses in wing disks and in cultured cells reveal that UbcD1 directly controls Ci stability. Interestingly, UbcD1 is found to be selectively involved in Slimb-mediated Ci degradation. Finally, we show that the homologs of UbcD1 play a conserved role in modulating Hh signaling in vertebrates.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Procesamiento Proteico-Postraduccional , Enzimas Ubiquitina-Conjugadoras/genética , Pez Cebra/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Proteínas Hedgehog/metabolismo , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Receptor Patched-2/genética , Receptor Patched-2/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Estabilidad Proteica , Proteolisis , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Although the RAG2 core domain is the minimal region required for V(D)J recombination, the noncore region also plays important roles in the regulation of recombination, and mutations in this region are often related to severe combined immunodeficiency. A complete understanding of the functions of the RAG2 noncore region and the potential contributions of its individual residues has not yet been achieved. Here, we show that the zinc finger motif within the noncore region of RAG2 is indispensable for maintaining the stability of the RAG2 protein. The zinc finger motif in the noncore region of RAG2 is highly conserved from zebrafish to humans. Knock-in mice carrying a zinc finger mutation (C478Y) exhibit decreased V(D)J recombination efficiency and serious impairment in T/B-cell development due to RAG2 instability. Further studies also reveal the importance of the zinc finger motif for RAG2 stability. Moreover, mice harboring a RAG2 noncore region mutation (N474S), which is located near C478 but is not zinc-binding, exhibit no impairment in either RAG2 stability or T/B-cell development. Taken together, our findings contribute to defining critical functions of the RAG2 zinc finger motif and provide insights into the relationships between the mutations within this motif and immunodeficiency diseases.
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Linfocitos B/inmunología , Proteínas de Unión al ADN/genética , Inmunodeficiencia Combinada Grave/genética , Linfocitos T/inmunología , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Secuencia de Bases , Células Cultivadas , Secuencia Conservada/genética , Técnicas de Inactivación de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Estabilidad Proteica , Alineación de Secuencia , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/citología , Recombinación V(D)J/genéticaRESUMEN
Photodynamic therapy (PDT) has emerged as a promising clinical modality for cancer therapy due to its ability to initiate an antitumor immune response. However, PDT-mediated cancer immunotherapy is severely impaired by tumor-cell immunosuppression of host T cell antitumor activity through the programmed cell death 1 ligand (PD-L1) and programmed cell death receptor 1 (PD-1) (PD-L1-PD-1) immune checkpoint pathway. Here, we demonstrate that PDT-mediated cancer immunotherapy can be augmented by PD-L1 knockdown (KD) in tumor cells. We rationally designed a versatile micelleplex by integrating an acid-activatable cationic micelle, photosensitizer (PS), and small interfering RNA (siRNA). The micelleplex was inert at physiological pH conditions and activated only upon internalization in the acidic endocytic vesicles of tumor cells for fluorescence imaging and PDT. Compared to PDT alone, the combination of PDT and PD-L1 KD showed significantly enhanced efficacy for inhibiting tumor growth and distant metastasis in a B16-F10 melanoma xenograft tumor model. These results suggest that acid-activatable micelleplexes utilizing PDT-induced cancer immunotherapy are more effective when combined with siRNA-mediated PD-L1 blockade. This study could provide a general strategy for enhancing the therapy efficacy of photodynamic cancer therapy.
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Inmunoterapia , Melanoma Experimental/tratamiento farmacológico , Fotoquimioterapia , Animales , Línea Celular Tumoral , Ratones , Ratones Endogámicos C57BL , Fármacos Fotosensibilizantes , Receptor de Muerte Celular Programada 1 , Linfocitos T , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Hippo signaling pathway has been identified to be involved in development and tissue homeostasis during the past decade, and is evolutionarily conserved from Drosophila to mammals. It transduces the signal through a series of protein-protein interaction and kinase cascades, to control the cell number and organ size by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo signaling pathway is associated with tumorigenesis and cancers, so it is a crucial target for cancer therapy and regeneration medicine. Most of the Hippo signaling pathway components have been identified, and the cellular function and molecular mechanism have been revealed by structural and functional researches. In this review, we summarize the molecular structure of Hippo signaling pathway components and related targeting inhibitors from a structural view. We hope to improve the understandings of the regulation mechanism of the Hippo signaling transduction, and facilitate further functional studies and potential therapeutic interventions.
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Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Ciclo Celular/química , Factor de Crecimiento de Hepatocito/química , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Estructura Molecular , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Serina-Treonina Quinasa 3 , Transactivadores , Factores de Transcripción/química , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Partial degradation of the p100 subunit to generate p52 subunit is a hallmark of the alternative NF-κB pathway, which has been implicated in cancer. Here, we uncovered a role of the p97-Npl4-Ufd1 complex in mediating p100-to-p52 processing and therefore positively regulating the alternative NF-κB pathway. We observed an elevation of p97 mRNA levels in lymphoma patients, which positively correlates with NFKB2 expression, a downstream target gene of the alternative NF-κB pathway. Moreover, NFKB2 mRNA levels were aberrantly down-regulated in patients with inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD), a disease caused by mutation of p97. Inactivation of p97 or depletion of the p97-Npl4-Ufd1 complex inhibits the processing of p100 into p52, decreasing transcription of the downstream target genes. Further analyses reveal that the p97-Npl4-Ufd1 complex interacts with F-box and WD repeats protein SCF(ßTrCP) complex to regulate the partial degradation of p100, a process involving K48- and K11-linked ubiquitination. In line with this, in LPS-induced lung damage mice model, generation of p52 is significantly decreased in p97-KD mice compared with mock mice. Finally, abrogation of p97 ATPase activity by its specific inhibitor DBeQ, efficiently decreased proliferation of lymphoma cells. Collectively, our study revealed a regulatory role of the p97-Npl4-Ufd1 complex in regulating p100 partial degradation, highlighting the potential of p97 as a drug target for cancers with aberrant activation of the alternative NF-κB pathway.