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1.
Nat Immunol ; 20(4): 458-470, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30890796

RESUMEN

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior activation via the T cell antigen receptor limits IL-6's control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4+ T cells. Transcriptomics and chromatin immunoprecipitation-sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4+ T cells sense and interpret cytokine signals.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Animales , Artritis Reumatoide/enzimología , Artritis Reumatoide/patología , Linfocitos T CD4-Positivos/enzimología , Células CHO , Células Cultivadas , Cricetulus , Regulación de la Expresión Génica , Humanos , Memoria Inmunológica , Interleucina-6/fisiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-6/fisiología , Membrana Sinovial/inmunología , Transcripción Genética
2.
Nature ; 627(8003): 437-444, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38383789

RESUMEN

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.


Asunto(s)
Retículo Endoplásmico , Procesamiento Proteico-Postraduccional , Subunidades Ribosómicas Grandes de Eucariotas , Ubiquitina-Proteína Ligasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , Microscopía por Crioelectrón , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Homeostasis , Membranas Intracelulares/metabolismo , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Peptidil Transferasas/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , ARN de Transferencia/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/metabolismo , Canales de Translocación SEC/ultraestructura , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/ultraestructura , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura
3.
EMBO J ; 41(21): e111015, 2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36121123

RESUMEN

Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold-type E3 ligase mechanism that activates the UFM1-conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.


Asunto(s)
Retículo Endoplásmico , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional , Estrés del Retículo Endoplásmico/fisiología , Unión Proteica , Proteínas Ribosómicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo
4.
J Immunol ; 211(2): 274-286, 2023 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-37272871

RESUMEN

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.


Asunto(s)
Interleucina-6 , Células TH1 , Animales , Ratones , Citocinas/metabolismo , Inflamación/metabolismo , Interleucina-6/metabolismo , Retroelementos , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Células TH1/metabolismo
5.
Proc Natl Acad Sci U S A ; 115(46): 11802-11807, 2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30373817

RESUMEN

Immunomodulatory drugs (IMiDs), including thalidomide derivatives such as lenalidomide and pomalidomide, offer therapeutic benefit in several hematopoietic malignancies and autoimmune/inflammatory diseases. However, it is difficult to study the IMiD mechanism of action in murine disease models because murine cereblon (CRBN), the substrate receptor for IMiD action, is resistant to some of IMiDs therapeutic effects. To overcome this difficulty, we generated humanized cereblon (CRBNI391V) mice thereby providing an animal model to unravel complex mechanisms of action in a murine physiological setup. In our current study, we investigated the degradative effect toward IKZF1 and CK-1α, a target substrate of IMiDs. Unlike WT mice which were resistant to lenalidomide and pomalidomide, T lymphocytes from CRBNI391V mice responded with a higher degree of IKZF1 and CK-1α protein degradation. Furthermore, IMiDs resulted in an increase in IL-2 among CRBNI391V mice but not in the WT group. We have also tested a thalidomide derivative, FPFT-2216, which showed an inhibitory effect toward IKZF1 protein level. As opposed to pomalidomide, FPFT-2216 and lenalidomide degrades CK-1α. Additionally, we assessed the potential therapeutic effects of IMiDs in dextran sodium sulfate (DSS)-induced colitis. In both WT and humanized mice, lenalidomide showed a significant therapeutic effect in the DSS model of colitis, while the effect of pomalidomide was less pronounced. Thus, while IMiDs' degradative effect on IKZF1 and CK-1α, and up-regulation of IL-2, is dependent on CRBN, the therapeutic benefit of IMiDs in a mouse model of inflammatory bowel disease occurs through a CRBN-IMiD binding region independent pathway.


Asunto(s)
Inmunomodulación/efectos de los fármacos , Inmunomodulación/fisiología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Humanos , Factor de Transcripción Ikaros/efectos de los fármacos , Factor de Transcripción Ikaros/metabolismo , Factores Inmunológicos/metabolismo , Ratones , Modelos Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/metabolismo
6.
Nucleic Acids Res ; 45(5): 2687-2703, 2017 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-28168301

RESUMEN

The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-6/genética , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Estabilidad del ARN , Receptor Toll-Like 4/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Animales , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(38): 10625-30, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27601648

RESUMEN

Immunomodulatory drugs (IMiDs) are a family of compounds derived from thalidomide. Binding of the IMiD molecule to the Lon protease Cereblon initiates the degradation of substrates via the ubiquitin proteasome pathway. Here, we show that Cereblon forms a complex with Rabex-5, a regulator of immune homeostasis. Treatment with lenalidomide prevented the association of Cereblon with Rabex-5. Conversely, mutation of the IMiD binding site increased Cereblon-Rabex-5 coimmunoprecipitation. The thalidomide binding region of Cereblon therefore regulates the formation of this complex. Knockdown of Rabex-5 in the THP-1 macrophage cell line up-regulated Toll-like receptor (TLR)-induced cytokine and type 1 IFN production via a STAT1/IRF activating pathway. Thus, we identify Rabex-5 as a IMiD target molecule that functions to restrain TLR activated auto-immune promoting pathways. We propose that release of Rabex-5 from complex with Cereblon enables the suppression of immune responses, contributing to the antiinflammatory properties of IMiDs.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Inmunidad Celular/genética , Interferón Tipo I/genética , Péptido Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Sitios de Unión/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Inmunidad Celular/efectos de los fármacos , Factores Inmunológicos/farmacología , Interferón Tipo I/biosíntesis , Lenalidomida , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/genética , Péptido Hidrolasas/genética , Unión Proteica/efectos de los fármacos , Talidomida/análogos & derivados , Talidomida/farmacología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Ubiquitina-Proteína Ligasas
8.
Int Immunol ; 29(2): 79-85, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28379390

RESUMEN

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are among the major causes of death worldwide due to acute inflammation in the lung. AT-rich interactive domain-containing 5a (Arid5a) is an RNA-binding protein involved in inflammatory autoimmune disease through post-transcriptional control of Il6, Stat3 and Tbx21 gene expression. We found that Arid5a-deficient mice were highly refractory to bleomycin (BLM)-induced lethality. Arid5a deficiency suppressed lung pathology, cytokine production (especially, IL-6), and clinical symptoms in BLM-treated mice. Production of reactive oxygen species (ROS) in response to BLM-induced cellular damage was inhibited in Arid5a-deficient mice, potentially affecting the level of oxidized 1-palmitoyl-2-arachidonoyl-phosphaticylcholine (OxPAPC) production. OxPAPC, which is supposed to be a TLR4/TLR2 ligand, stimulated expression of the Arid5a and Il6 genes. Thus, reduction of ROS production in Arid5a-deficient mice could mitigate OxPAPC production, which in turn decreases IL-6 production in vivo due to dysregulated post-transcriptional regulation by loss of Arid5a. Therefore, the control of Arid5a expression represents a potential therapeutic target for treatment of ALI and ARDS.


Asunto(s)
Lesión Pulmonar Aguda/inmunología , Proteínas de Unión al ADN/genética , Pulmón/patología , Neumonía/inmunología , Síndrome de Dificultad Respiratoria/inmunología , Factores de Transcripción/genética , Lesión Pulmonar Aguda/inducido químicamente , Animales , Bleomicina/administración & dosificación , Humanos , Interleucina-6/metabolismo , Pulmón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/terapia , Especies Reactivas de Oxígeno/metabolismo , Síndrome de Dificultad Respiratoria/terapia
9.
Int Immunol ; 28(6): 307-15, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26865412

RESUMEN

Thalidomide and its derivatives, collectively referred to as immunomodulatory drugs (IMiDs), are effective inhibitors of inflammation and are known to inhibit TLR-induced TNFα production. The identification of Cereblon as the receptor for these compounds has led to a rapid advancement in our understanding of IMiD properties; however, there remain no studies addressing the role of Cereblon in mediating the suppressive effect of IMiDs on TLR responses. Here, we developed Cereblon-deficient mice using the CRISPR-Cas9 system. TLR-induced cytokine responses were unaffected by Cereblon deficiency in vivo Moreover, IMiD treatment inhibited cytokine production even in the absence of Cereblon. The IMiD-induced suppression of cytokine production therefore occurs independently of Cereblon in mice. Further investigation revealed that IMiDs are potent inhibitors of TLR-induced type-1 interferon production via suppression of the TRIF/IRF3 pathway. These data suggest that IMiDs may prove effective in the treatment of disorders characterized by the ectopic production of type-1 interferon. Significantly, these properties are mediated separately from thalidomide's teratogenic receptor, Cereblon. Thus, certain therapeutic properties of Thalidomide can be separated from its harmful side effects.


Asunto(s)
Factores Inmunológicos/uso terapéutico , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Talidomida/análogos & derivados , Talidomida/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Terapia de Inmunosupresión , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Lenalidomida , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
10.
Int Immunol ; 27(8): 405-15, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25862525

RESUMEN

Aryl hydrocarbon receptor (Ahr), a transcription factor, plays a critical role in autoimmune inflammation of the intestine. In addition, microRNAs (miRNAs), small non-coding oligonucleotides, mediate pathogenesis of inflammatory bowel diseases (IBD). However, the precise mechanism and interactions of these molecules in IBD pathogenesis have not yet been investigated. We analyzed the role of Ahr and Ahr-regulated miRNAs in colonic inflammation. Our results show that deficiency of Ahr in intestinal epithelial cells in mice exacerbated inflammation in dextran sodium sulfate-induced colitis. Deletion of Ahr in T cells attenuated colitis, which was manifested by suppressed Th17 cell infiltration into the lamina propria. Candidate miRNA analysis showed that induction of colitis elevated expression of the miR-212/132 cluster in the colon of wild-type mice, whereas in Ahr (-/-) mice, expression was clearly lower. Furthermore, miR-212/132(-/-) mice were highly resistant to colitis and had reduced levels of Th17 cells and elevated levels of IL-10-producing CD4(+) cells. In vitro analyses revealed that induction of type 1 regulatory T (Tr1) cells was significantly elevated in miR-212/132(-/-) T cells with increased c-Maf expression. Our findings emphasize the vital role of Ahr in intestinal homeostasis and suggest that inhibition of miR-212/132 represents a viable therapeutic strategy for treating colitis.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Colitis/genética , Interleucina-10/genética , MicroARNs/genética , Receptores de Hidrocarburo de Aril/genética , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Proliferación Celular , Colitis/inducido químicamente , Colitis/inmunología , Colitis/patología , Sulfato de Dextran , Femenino , Regulación de la Expresión Génica , Homeostasis/inmunología , Interleucina-10/inmunología , Intestinos/inmunología , Intestinos/patología , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/inmunología , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-maf/inmunología , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/inmunología , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th17/inmunología , Células Th17/patología
11.
Proc Natl Acad Sci U S A ; 110(29): 11964-9, 2013 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-23818645

RESUMEN

Aryl hydrocarbon receptor (AHR) plays critical roles in various autoimmune diseases such as multiple sclerosis by controlling interleukin-17 (IL-17)-producing T-helper (TH17) and regulatory T cells. Although various transcription factors and cytokines have been identified as key participants in TH17 generation, the role of microRNAs in this process is poorly understood. In this study, we found that expression of the microRNA (miR)-132/212 cluster is up-regulated by AHR activation under TH17-inducing, but not regulatory T-inducing conditions. Deficiency of the miR-132/212 cluster prevented the enhancement of TH17 differentiation by AHR activation. We also identified B-cell lymphoma 6, a negative regulator of TH17 differentiation, as a potential target of the miR-212. Finally, we investigated the roles of the miR-132/212 cluster in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. Mice deficient in the miR-132/212 cluster exhibited significantly higher resistance to the development of experimental autoimmune encephalomyelitis and lower frequencies of both TH1 and TH17 cells in draining lymph nodes. Our findings reveal a unique mechanism of AHR-dependent TH17 differentiation that depends on the miR-132/212 cluster.


Asunto(s)
Diferenciación Celular/inmunología , Interleucina-17/metabolismo , MicroARNs/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Western Blotting , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Interleucina-17/inmunología , Luciferasas , Ratones , Ratones Noqueados , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Colaboradores-Inductores/metabolismo
12.
Biochem Biophys Res Commun ; 456(1): 527-33, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25490391

RESUMEN

Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.


Asunto(s)
Anticuerpos Monoclonales/química , Inmunohistoquímica , Proteínas/metabolismo , Proteínas Recombinantes/química , Animales , Antígenos/inmunología , Membrana Celular/metabolismo , Epítopos/inmunología , Hibridomas/inmunología , Ratones , Neuronas/metabolismo , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Pez Cebra
13.
FEBS J ; 290(21): 5040-5056, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-36680403

RESUMEN

Ubiquitin Fold Modifier-1 (UFM1) is a ubiquitin-like modifier (UBL) that is posttranslationally attached to lysine residues on substrates via a dedicated system of enzymes conserved in most eukaryotes. Despite the structural similarity between UFM1 and ubiquitin, the UFMylation machinery employs unique mechanisms that ensure fidelity. While physiological triggers and consequences of UFMylation are not entirely clear, its biological importance is epitomized by mutations in the UFMylation pathway in human pathophysiology including musculoskeletal and neurodevelopmental diseases. Some of these diseases can be explained by the increased endoplasmic reticulum (ER) stress and disrupted translational homeostasis observed upon loss of UFMylation. The roles of UFM1 in these processes likely stem from its function at the ER where ribosomes are UFMylated in response to translational stalling. In addition, UFMylation has been implicated in other cellular processes including DNA damage response and telomere maintenance. Hence, the study of UFM1 pathway mechanics and its biological function will reveal insights into fundamental cell biology and is likely to afford new therapeutic opportunities for the benefit of human health. To this end, we herein provide a comprehensive guide to the current state of knowledge of UFM1 biogenesis, conjugation, and function with an emphasis on the underlying mechanisms.


Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas , Humanos , Proteínas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo
14.
Methods Mol Biol ; 2691: 81-95, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37355539

RESUMEN

Antimicrobial host defense is dependent on the rapid recruitment of inflammatory cells to the site of infection, the elimination of invading pathogens, and the efficient resolution of inflammation that minimizes damage to the host. The peritoneal cavity provides an accessible and physiologically relevant system where the delicate balance of these processes may be studied. Here, we describe murine models of peritoneal inflammation that enable studies of competent antimicrobial immunity and inflammation-associated tissue damage as a consequence of recurrent bacterial challenge. The inflammatory hallmarks of these models reflect the clinical and molecular features of peritonitis seen in renal failure patients on peritoneal dialysis. The development of these models relies on the preparation of a cell-free supernatant derived from an isolate of Staphylococcus epidermidis (termed SES). Intraperitoneal administration of SES induces a Toll-like receptor 2-driven acute inflammatory response that is characterized by an initial transient influx of neutrophils that are replaced by a more sustained recruitment of mononuclear cells and lymphocytes. Adaptation of this model using a repeated administration of SES allows investigations into the development of adaptive immunity and the hallmarks associated with tissue remodelling and fibrosis. These models are therefore clinically relevant and provide exciting opportunities to study innate and adaptive immunity and the response of the stromal tissue compartment to bacterial infection and the ensuing inflammatory reaction.


Asunto(s)
Diálisis Peritoneal , Peritonitis , Humanos , Ratones , Animales , Peritoneo , Inflamación , Cavidad Peritoneal , Diálisis Peritoneal/efectos adversos
15.
FEBS Lett ; 596(5): 567-588, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34618359

RESUMEN

Unravelling the molecular mechanisms that account for functional pleiotropy is a major challenge for researchers in cytokine biology. Cytokine-receptor cross-reactivity and shared signalling pathways are considered primary drivers of cytokine pleiotropy. However, reports epitomized by studies of Jak-STAT cytokine signalling identify interesting biochemical and epigenetic determinants of transcription factor regulation that affect the delivery of signal-dependent cytokine responses. Here, a regulatory interplay between STAT transcription factors and their convergence to specific genomic enhancers support the fine-tuning of cytokine responses controlling host immunity, functional identity, and tissue homeostasis and repair. In this review, we provide an overview of the signalling networks that shape the way cells sense and interpret cytokine cues. With an emphasis on the biology of interleukin-6, we highlight the importance of these mechanisms to both physiological processes and pathophysiological outcomes.


Asunto(s)
Señales (Psicología) , Interleucina-6 , Citocinas/metabolismo , Interleucina-6/metabolismo , Quinasas Janus/genética , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de Señal
16.
Cell Rep ; 40(5): 111168, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35926457

RESUMEN

An essential first step in the post-translational modification of proteins with UFM1, UFMylation, is the proteolytic cleavage of pro-UFM1 to expose a C-terminal glycine. Of the two UFM1-specific proteases (UFSPs) identified in humans, only UFSP2 is reported to be active, since the annotated sequence of UFSP1 lacks critical catalytic residues. Nonetheless, efficient UFM1 maturation occurs in cells lacking UFSP2, suggesting the presence of another active protease. We herein identify UFSP1 translated from a non-canonical start site to be this protease. Cells lacking both UFSPs show complete loss of UFMylation resulting from an absence of mature UFM1. While UFSP2, but not UFSP1, removes UFM1 from the ribosomal subunit RPL26, UFSP1 acts earlier in the pathway to mature UFM1 and cleave a potential autoinhibitory modification on UFC1, thereby controlling activation of UFMylation. In summary, our studies reveal important distinctions in substrate specificity and localization-dependent functions for the two proteases in regulating UFMylation.


Asunto(s)
Péptido Hidrolasas , Procesamiento Proteico-Postraduccional , Humanos , Cisteína Endopeptidasas/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Especificidad por Sustrato
17.
Methods Mol Biol ; 1725: 65-75, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29322409

RESUMEN

Anti-microbial host defence is dependent on the rapid recruitment of inflammatory cells to the site of infection, the elimination of invading pathogens, and the efficient resolution of inflammation so as to minimise damage to the host. The peritoneal cavity provides an easily accessible and physiologically relevant system where the delicate balance of these processes may be studied. Here, we describe murine models of peritoneal inflammation that enable studies of both competent anti-microbial immunity and inflammation associated tissue damage as a consequence of recurrent bacterial challenge. The inflammatory hallmarks of these models reflect the clinical and molecular features of peritonitis episodes seen in renal failure patients on peritoneal dialysis. Development of these models relies on the preparation of a cell-free supernatant derived from an isolate of Staphylococcus epidermidis (termed SES). Intraperitoneal administration of SES induces a TLR2-driven acute inflammatory response that is characterised by an initial transient influx of neutrophils that are replaced by a more sustained recruitment of mononuclear cells and lymphocytes. Adaptation of this model using a repeated administration of SES allows investigations into the development of adaptive immunity and memory responses, and the hallmarks associated with tissue remodelling and fibrosis. These models are therefore clinically relevant and provide exciting opportunities to study both innate and adaptive immune responses in the control of bacterial infection and pathogenesis.


Asunto(s)
Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Inflamación/etiología , Macrófagos Peritoneales/inmunología , Peritonitis/etiología , Infecciones Estafilocócicas/complicaciones , Staphylococcus epidermidis/aislamiento & purificación , Inmunidad Adaptativa , Animales , Enfermedad Crónica , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Peritonitis/metabolismo , Peritonitis/patología , Infecciones Estafilocócicas/microbiología
18.
Trends Mol Med ; 23(4): 348-361, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28285807

RESUMEN

Thalidomide and its derivatives are immunomodulatory drugs (IMiDs) known for their sedative, teratogenic, anti-angiogenic, and anti-inflammatory properties. Commonly used in the treatment of cancers such as multiple myeloma and myelodysplastic syndrome (MDS), IMiDs have also been used in the treatment of an inflammatory skin pathology associated with Hansen's disease/leprosy. They have also shown promise in the treatment of autoimmune disorders including systemic lupus erythmatosus (SLE) and inflammatory bowel disease (IBD). Recent structural and experimental observations have revolutionized our understanding of these properties by revealing the fundamental molecular events underpinning IMiD activity. We review these findings, their relevance to IMiD therapy in immunological disorders, and discuss how further research might unlock the vast clinical potential of these compounds.


Asunto(s)
Antiinflamatorios/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Lepra/tratamiento farmacológico , Talidomida/uso terapéutico , Animales , Antiinflamatorios/farmacología , Enfermedades Autoinmunes/inmunología , Humanos , Lepra/inmunología , Modelos Moleculares , Talidomida/farmacología
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