RESUMEN
A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.
Asunto(s)
Complejo CD3/metabolismo , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Complejo CD3/química , Proteína Tirosina Quinasa CSK/metabolismo , Línea Celular , Citocinas/metabolismo , Humanos , Activación de Linfocitos/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Ratones Endogámicos NOD , Neoplasias/mortalidad , Neoplasias/patología , Neoplasias/terapia , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Supervivencia , Vanadatos/farmacologíaRESUMEN
The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.
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Nucléolo Celular , Exosomas , Precursores del ARN , Procesamiento Postranscripcional del ARN , ARN Ribosómico , Pez Cebra , Animales , Ratones , Nucléolo Celular/metabolismo , Desarrollo Embrionario , Exosomas/metabolismo , Cabeza/anomalías , Microscopía , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 28S/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
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Caveolina 1 , Movimiento Celular , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Transporte Biológico , Cicatrización de Heridas/fisiología , Orgánulos/metabolismoRESUMEN
The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.
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Exosomas/genética , Exosomas/metabolismo , Proteínas Nucleares/fisiología , ARN Helicasas/metabolismo , Animales , Núcleo Celular/metabolismo , Células Madre Embrionarias , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas Nucleares/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas/genética , Estabilidad del ARN/genéticaRESUMEN
Oxidative stress is a key feature in both chronic inflammation and cancer. P38 regulated/activated protein kinase (PRAK) deficiency can cause functional disorders in neutrophils and macrophages under high oxidative stress, but the precise mechanisms by which PRAK regulates reactive oxygen species (ROS) elimination and its potential impact on CD4+ T helper subset function are unclear. The present study reveals that the PRAK-NF-E2-related factor 2(NRF2) axis is essential for maintaining the intracellular redox homeostasis of T helper 17(Th17) cells, thereby promoting Th17 cell differentiation and antitumor effects. Through mechanistic analysis, we identify NRF2 as a novel protein substrate of PRAK and find that PRAK enhances the stability of the NRF2 protein through phosphorylation NRF2 Serine(S) 558 independent of protein ubiquitination. High accumulation of cellular ROS caused by loss of PRAK disrupts both glycolysis and PKM2-dependent phosphorylation of STAT3, which subsequently impairs the differentiation of Th17 cells. As a result, Prak knockout (KO) mice display significant resistance to experimental autoimmune encephalomyelitis (EAE) but impaired antitumor immunity in a MC38 tumor model. This work reveals that the PRAK-NRF2-mediated antioxidant pathway is a metabolic checkpoint that controls Th17-cell glycolysis and differentiation. Targeting PRAK is a promising strategy for maintaining an active ROS scavenging system and may lead to potent Th17 cell antitumor immunity.
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Encefalomielitis Autoinmune Experimental , Proteínas Quinasas , Animales , Ratones , Diferenciación Celular , Glucólisis , Homeostasis , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Th17/metabolismoRESUMEN
Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.
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Neoplasias Colorrectales , Histonas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteómica , Humanos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Proteómica/métodos , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Histonas/metabolismo , Transglutaminasas/metabolismo , Transglutaminasas/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Línea Celular Tumoral , Proteoma/análisis , Proteoma/metabolismo , Procesamiento Proteico-Postraduccional , Glutamina/metabolismo , Glutamina/química , Epigénesis GenéticaRESUMEN
Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.
RESUMEN
The COVID-19 pandemic has become a worldwide health crisis. So far, most studies have focused on the epidemiology and pathogenesis of this infectious disease. Little attention has been given to the disease sequelae in patients recovering from COVID-19, and nothing is known about the mechanisms underlying these sequelae. Herein, we profiled the serum proteome of a cohort of COVID-19 patients in the disease onset and recovery stages. Based on the close integration of our proteomic analysis with clinical data, we propose that COVID-19 is associated with prolonged disorders in cholesterol metabolism and myocardium, even in the recovery stage. We identify potential biomarkers for these disorders. Moreover, severely affected patients presented more serious disturbances in these pathways. Our findings potentially support clinical decision-making to improve the prognosis and treatment of patients.
Asunto(s)
COVID-19 , Proteómica , Colesterol , Humanos , Miocardio , Pandemias , Proteoma , SARS-CoV-2RESUMEN
BACKGROUND: Preoperative treatment of anti-vascular endothelial growth factor (VEGF) agents is extensively used in proliferative diabetic retinopathy (PDR), but the molecular mechanism is not fully understood. The objective of this research is to observe change of protein profile induced by ranibizumab (an anti-VEGF agent) in vitreous humor from PDR patients and reveal the effects of anti-VEGF treatment on PDR. METHODS: A proteomic method was used to identify differentially expressed proteins in vitreous humor. Untreated PDR patients were defined as PDR group, while those who treated with intravitreal injection of ranibizumab (IVR) were defined as IVR. Gene Ontology (GO) annotation and REACTOME pathways were obtained from DAVID Bioinformatics Resources. Intravitreal level of apolipoprotein C-I (APOC1), serpin peptidase inhibitor clade A member 5 (SERPINA5), tissue inhibitor of metalloproteinases (TIMP2), and keratin 1 (KRT1) were determined by enzyme-linked immuno sorbent assay (ELISA). RESULTS: 339 differentially expressed proteins were identified in response to IVR. The most notable GO annotation describes the altered proteins was "innate immune response". The most notable REACTOME pathway was "platelet degranulation". ELISA result showed increased level of APOC1, SERPINA5, KRT1 and a decreased level of TIMP2 in PDR group compared with IVR. CONCLUSIONS: In addition to decreasing VEGF level, ranibizumab is associated with change of human vitreous protein profile in patients with PDR, in which the differential proteins are involved in immune response, platelet degranulation, complement activation etc., suggesting that the effects of VEGF are involved in these signaling pathways.
RESUMEN
Advanced-stage cutaneous T-cell lymphomas (CTCLs) are notorious for their highly aggressive behavior, resistance to conventional treatments, and poor prognosis, particularly when large-cell transformation occurs. PEG10 has been recently proposed as a potent driver for large-cell transformation in CTCL. However, the targeting of PEG10 continues to present a formidable clinical challenge that has yet to be addressed. In this study, we report an important post-translational regulatory mechanism of PEG10 in CTCL. USP9X, a deubiquitinase, interacted with and deubiquitinated PEG10, thereby stabilizing PEG10. Knockdown of USP9X or pharmacological targeting of USP9X resulted in a prominent downregulation of PEG10 and its downstream pathway in CTCL. Moreover, USP9X inhibition conferred tumor cell growth disadvantage and enhanced apoptosis in vitro, an effect that occurred in part through its regulation on PEG10. Furthermore, we demonstrated that inhibition of USP9X obviously restrained CTCL tumor growth in vivo and that high expression of USP9X is associated with poor survival in patients with CTCL. Collectively, our findings uncover USP9X as a key post-translational regulator in the stabilization of PEG10 and suggest that targeting PEG10 stabilization through USP9X inhibition may represent a promising therapeutic strategy for advanced-stage CTCL.
RESUMEN
Serotonylation has been identified as a novel protein post-translational modification (PTM) for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibody for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to label protein serotonylation in a bioorthogonal manner. This chemical biology tool can be utilized alternatively for the antibody-free enrichment of serotonylated proteins, which depends on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 500 serotonylated proteins were identified from HCT 116 cells. Importantly, a number of modification sites of these serotonylated proteins were determine, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.
RESUMEN
Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 ( i.e. , glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy.
RESUMEN
Histone monoaminylation ( i . e ., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.
RESUMEN
Vγ9Vδ2 T cells are specialized effector cells that have gained prominence as immunotherapy agents due to their ability to target and kill cells with altered pyrophosphate metabolites. In our effort to understand how cancer cells evade the cell-killing activity of Vγ9Vδ2 T cells, we performed a comprehensive genome-scale CRISPR screening of cancer cells. We found that four molecules belonging to the butyrophilin (BTN) family, specifically BTN2A1, BTN3A1, BTN3A2, and BTN3A3, are critically important and play unique, nonoverlapping roles in facilitating the destruction of cancer cells by primary Vγ9Vδ2 T cells. The coordinated function of these BTN molecules was driven by synchronized gene expression, which was regulated by IFN-γ signaling and the RFX complex. Additionally, an enzyme called QPCTL was shown to play a key role in modifying the N-terminal glutamine of these BTN proteins and was found to be a crucial factor in Vγ9Vδ2 T cell killing of cancer cells. Through our research, we offer a detailed overview of the functional genomic mechanisms that underlie how cancer cells escape Vγ9Vδ2 T cells. Moreover, our findings shed light on the importance of the harmonized expression and function of gene family members in modulating T-cell activity.
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Neoplasias , Linfocitos T , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Butirofilinas/genética , Butirofilinas/metabolismo , Activación de Linfocitos/genética , Muerte CelularRESUMEN
The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.
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ADN Helicasas , ARN Helicasas , ADN Helicasas/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Separación de Fases , Gránulos Citoplasmáticos/metabolismoRESUMEN
A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos+ neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors.Abbreviation: AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine-N-oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element.
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Ansiedad , Macroautofagia , Neuralgia , Neuronas , Corteza Prefrontal , Animales , Neuralgia/metabolismo , Corteza Prefrontal/metabolismo , Ratas , Neuronas/metabolismo , Masculino , Macroautofagia/fisiología , Ratas Sprague-Dawley , Conducta Animal , Dolor Crónico/metabolismo , Autofagia/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , HiperalgesiaRESUMEN
SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 Å. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3. ARM1 is in the inhibited state with the active site of Rpd3 blocked, whereas ARM2 is in an open conformation with the active site of Rpd3 exposed to the exterior space. The observed asymmetric architecture of Rpd3L is different from those of available structures of other class I HDAC complexes. Our study reveals the organization mechanism of the SIN3-HDAC complex and provides insights into the interaction pattern by which it targets histone deacetylase to chromatin.
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Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Factores de Transcripción/metabolismo , Proteínas Represoras/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina , Histona Desacetilasas/genéticaRESUMEN
Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.
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Autofagosomas , Filaminas , Macroautofagia , Proteínas Qa-SNARE , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Fosforilación , Humanos , Proteínas Qa-SNARE/metabolismo , Filaminas/metabolismoRESUMEN
Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.
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Lubina , Iridovirus , Ranavirus , Animales , Iridovirus/metabolismo , Proteínas de la Cápside/metabolismo , Singapur , Ranavirus/metabolismo , Ensamble de VirusRESUMEN
Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.