RESUMEN
Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.
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Glucosilceramidas , Virus ARN , Glucosilceramidas/metabolismo , Fusión de Membrana , Glicoproteínas/química , Lactosilceramidos , Virus ARN/metabolismoRESUMEN
UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.
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Autofagia/efectos de la radiación , Daño del ADN , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Melanoma Experimental/enzimología , Neoplasias Cutáneas/enzimología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Rayos Ultravioleta , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Activación Enzimática , Células HEK293 , Células HeLa , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Proteolisis , Interferencia de ARN , Retina/enzimología , Retina/efectos de la radiación , Transducción de Señal/efectos de la radiación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
OBJECTIVES: This up-to-date systematic review and meta-analysis aimed to examine the predictive validity of the Geriatric Depression Scale-15 (GDS-15) for screening depression in older adults aged over 65 years. METHODS: Electronic searches were performed on the MEDLINE, EMBASE, CINAHL, and PsycINFO database using the following keywords: depression, depressive disorder, major, geriatric depression scale, and geriatric depression scale short. The Quality Assessment of Diagnostic Accuracy Studies-2 was applied to assess the risk of bias in diagnostic studies. RESULTS: Thirty-one studies that included 8,897 older adults were analyzed. The pooled sensitivity of the GDS-15 was 0.80 (95% CI:0.78 to 0.82), its pooled specificity was 0.79 (95% CI:0.78 to 0.80), the area under the curve (AUC) was 0.89 (SE = 0.01) and the Q* value was 0.82 (SE = 0.01). The subgroup analysis revealed that the pooled sensitivity and specificity of the GDS-15 were higher in older adults with normal cognitive function than in those with cognitive impairment. CONCLUSIONS: These finding suggest that the GDS-15 may be more accurate for screening depression in older adults with normal cognitive function. CLINICAL IMPLICATIONS: The utility the GDS-15 may be restricted because its diagnostic accuracy is slightly lower among older adults with cognitive impairment.
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Depresión , Evaluación Geriátrica , Anciano , Depresión/diagnóstico , Humanos , Tamizaje Masivo , Escalas de Valoración Psiquiátrica , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: This study aims to increase understanding of how patient and family education affects the prevention of medical errors, thereby providing basic data for developing educational contents. METHODS: This descriptive study surveyed patients, families, and Patient Safety Officers to investigate the relationship between educational contents and medical error prevention. The Chi-square test and ANOVA were used to derive the results of this study. The educational contents used in this study consisted of health information (1. current medicines, 2. allergies, 3. health history, 4. previous treatments/tests and complications associated with them) and Speak Up (1. handwashing, 2. patient identification, 3. asking about medical conditions, 4. asking about test results, 5. asking about behaviour and changes in lifestyle, 6. asking about the care plan, 7. asking about medicines, and 8. asking about medicine interactions). RESULTS: In this study, the first criterion for choosing a hospital for treatment in Korea was 'Hospital with a famous doctor' (58.6% patient; 57.7% families). Of the patients and their families surveyed, 82.2% responded that hospitals in Korea were safe. The most common education in hospitals is 'Describe your medical condition', given to 69.0% of patients, and 'Hospitalisation orientation', given to 63.4% of families. The most important factors in preventing patient safety events were statistically significant differences among patients, family members, and Patient Safety Officers (p = 0.001). Patients and families had the highest 'Patient and family participation' (31.0% of patients; 39.4% of families) and Patient Safety Officers had the highest 'Patient safety culture' (47.8%). CONCLUSIONS: Participants thought that educational contents developed through this study could prevent medical errors. The results of this study are expected to provide basic data for national patient safety campaigns and standardised educational content development to prevent medical errors.
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Familia , Educación en Salud , Errores Médicos/prevención & control , Educación del Paciente como Asunto , Adulto , Encuestas de Atención de la Salud , Hospitales , Humanos , Persona de Mediana Edad , Seguridad del Paciente , República de Corea , Administración de la SeguridadRESUMEN
The cullin-RING ubiquitin ligases are multisubunit complexes that ubiquitinate various proteins. Six different cullins encoded by the human genome selectively pair with different adaptors and substrate receptors. It is presently poorly understood how cullin-2 (Cul2) and cullin-5 (Cul5) associate specifically with their adaptor elongin BC and a SOCS-box-containing substrate receptor. Here, crystallographic and mutational analyses of a quaternary complex between the N-terminal half of Cul5, elongin BC and SOCS2 are reported. Cul5 interacts extensively with elongin BC via residues that are highly conserved in Cul2 but not in other cullins. Cul5 also interacts with SOCS2, but via only two residues, Pro184 and Arg186, which are located in the C-terminal part of the SOCS box called the Cul5 box. Pro184 makes a ring-to-ring interaction with Trp53 of Cul5, which is substituted by alanine in Cul2. This interaction is shown to contribute significantly to the overall binding affinity between Cul5 and SOCS2-elongin BC. This study provides structural bases underlying the specificity of Cul5 and Cul2 for elongin BC and their preferential association with Cul5 or Cul2 box-containing substrate receptors.
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Proteínas Cullin/química , Proteínas Supresoras de la Señalización de Citocinas/química , Factores de Transcripción/química , Arginina , Calorimetría/métodos , Cristalografía por Rayos X , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Elonguina , Humanos , Modelos Moleculares , Prolina , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.
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COVID-19 , VIH-1 , Humanos , SARS-CoV-2/genética , Exorribonucleasas/genética , VIH-1/genética , Simulación del Acoplamiento Molecular , Antivirales/farmacología , Replicación Viral , Catecoles/farmacología , Ribonucleasa H/farmacología , Proteínas no Estructurales Virales/genética , ARN Viral/genéticaRESUMEN
Dabie Bandavirus (DBV), previously known as Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus, induces a characteristic thrombocytopenia with a mortality rate ranging from 12% to as high as 30%. The sero-prevalence of DBV in healthy people is not significantly different among age groups, but clinically diagnosed SFTS patients are older than ~50 years, suggesting that age is the critical risk factor for SFTS morbidity and mortality. Accordingly, our immune-competent ferret model demonstrates an age (>4 years old)-dependent DBV infection and pathogenesis that fully recapitulates human clinical manifestation. To protect the aged population from DBV-induced SFTS, vaccine should carry robust immunogenicity with high safety profile. Previous studies have shown that glycoproteins Gn/Gc are the most effective antigens for inducing both neutralizing antibody (NAb)- and T cell-mediated immunity and, thereby, protection. Here, we report the development of a protein subunit vaccine with 24-mer self-assembling ferritin (FT) nanoparticle to present DBV Gn head region (GnH) for enhanced immunogenicity. Anion exchange chromatography and size exclusion chromatography readily purified the GnH-FT nanoparticles to homogeneity with structural integrity. Mice immunized with GnH-FT nanoparticles induced robust NAb response and T-cell immunity against DBV Gn. Furthermore, aged ferrets immunized with GnH-FT nanoparticles were fully protected from DBV challenge without SFTS symptoms such as body weight loss, thrombocytopenia, leukopenia, and fatality. This study demonstrates that DBV GnH-FT nanoparticles provide an efficient vaccine efficacy in mouse and aged ferret models and should be an outstanding vaccine candidate targeted for the aged population against fatal DBV infection.
RESUMEN
IMPORTANCE: Dabie bandavirus (DBV) is an emerging tick-borne virus that causes severe fever with thrombocytopenia syndrome (SFTS) in infected patients. Human SFTS symptoms progress from fever, fatigue, and muscle pain to the depletion of white blood cells and platelets with fatality rates up to 30%. The recent spread of its vector tick to over 20 states in the United States increases the potential for outbreaks of the SFTS beyond the East Asia. Thus, the development of vaccine to control this rapidly emerging virus is a high priority. In this study, we applied self-assembling ferritin (FT) nanoparticle to enhance the immunogenicity of DBV Gn head domain (GnH) as a vaccine target. Mice immunized with the GnH-FT nanoparticle vaccine induced potent antibody responses and cellular immunity. Immunized aged ferrets were fully protected from the lethal challenge of DBV. Our study describes the GnH-FT nanoparticle vaccine candidate that provides protective immunity against the emerging DBV infection.
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Hurones , Síndrome de Trombocitopenia Febril Grave , Humanos , Animales , Ratones , Anciano , Nanovacunas , Modelos Animales de Enfermedad , FerritinasRESUMEN
Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.
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Asma , Factor de Células Madre , Animales , Ratones , Proliferación Celular , Pulmón/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismoRESUMEN
With the emergence of multiple predominant SARS-CoV-2 variants, it becomes important to have a comprehensive assessment of their viral fitness and transmissibility. Here, we demonstrate that natural temperature differences between the upper (33°C) and lower (37°C) respiratory tract have profound effects on SARS-CoV-2 replication and transmissibility. Specifically, SARS-CoV-2 variants containing the NSP12 mutations P323L or P323L/G671S exhibit enhanced RNA-dependent RNA polymerase (RdRp) activity at 33°C compared with 37°C and high transmissibility. Molecular dynamics simulations and microscale thermophoresis demonstrate that the NSP12 P323L and P323L/G671S mutations stabilize the NSP12-NSP7-NSP8 complex through hydrophobic effects, leading to increased viral RdRp activity. Furthermore, competitive transmissibility assay reveals that reverse genetic (RG)-P323L or RG-P323L/G671S NSP12 outcompetes RG-WT (wild-type) NSP12 for replication in the upper respiratory tract, allowing markedly rapid transmissibility. This suggests that NSP12 P323L or P323L/G671S mutation of SARS-CoV-2 is associated with increased RdRp complex stability and enzymatic activity, promoting efficient transmissibility.
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COVID-19 , SARS-CoV-2 , Animales , Humanos , SARS-CoV-2/genética , Hurones , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/química , Mutación/genética , Replicación Viral/genéticaRESUMEN
SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc-ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc-ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.
RESUMEN
The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Tipo IV/química , Sistemas de Secreción Tipo IV/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Línea Celular , Cristalografía por Rayos X , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Unión Proteica , Transporte de Proteínas , Sistemas de Secreción Tipo IV/genéticaRESUMEN
The aim of this comparative study involving pre- and post-tests was to analyze the effectiveness of patient safety educational materials developed for the Comprehensive Plans for Patient Safety in Korea (2018-2022), and to suggest how to improve patient safety literacy. A face-to-face survey interview comprising items related to general information and patient safety literacy was completed by 217 patients and their families who visited three general hospitals in Seoul and one general hospital in Gyeonggi-do for treatment between 25 October and 15 November 2019. In the interview, the patients were asked questions about whether the patient safety educational materials were "easy to understand," provided "help in safe hospitalization," and enabled patients to practice patient safety independently ("do it yourself"). The literacy of the patient safety educational materials was analyzed using a paired t-test with a p value of 0.05. The comparison between patient safety literacy on pre- and post-tests revealed that among all participants, there were significant differences in "easy to understand," "help in safe hospitalization," and "do it yourself" scores. To improve patient safety literacy, patient education materials need to optimize communication by improving patients' knowledge, skills, and attitudes for maintaining and promoting healthy living.
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Conocimientos, Actitudes y Práctica en Salud , Alfabetización en Salud/métodos , Educación del Paciente como Asunto/métodos , Seguridad del Paciente , Adulto , Comunicación , Comprensión , Femenino , Humanos , Masculino , Persona de Mediana Edad , República de Corea , SeúlRESUMEN
BACKGROUND: Replacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. OBJECTIVES: To reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti-tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. METHODS: MG1113 is an IgG4 monoclonal antibody that binds to Kunitz-2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)- or IX (FIX)-deficient plasma. For the ex vivo spiking test, platelet-poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)-induced rabbits. RESULTS: Radiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII-deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration-dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113-treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA-induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. CONCLUSIONS: These results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.
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Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.
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Receptores de Antígenos de Linfocitos T/química , Receptores Quiméricos de Antígenos/química , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/efectos de los fármacos , Ingeniería Celular , Células Cultivadas , Humanos , Inmunoterapia Adoptiva , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Células PC-3 , Unión Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/antagonistas & inhibidores , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Many bacteria, including Legionella pneumophila, rely on the type IV secretion system to translocate a repertoire of effector proteins into the hosts for their survival and growth. Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating substrates to the transenvelope secretion conduit. Yet, how a large number of effector proteins are selectively recruited and processed by T4CPs remains enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain and a C-terminal extension whose function is unknown. Unlike T4CPs involved in plasmid DNA translocation, DotL appeared to function by forming a multiprotein complex with four other proteins. Here, we show that the C-terminal extension of DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA, and that this pentameric assembly binds Legionella effector proteins. We determined the crystal structure of this assembly and built an architecture of the T4CP holocomplex by combining a homology model of the ATPase domain of DotL. The holocomplex is a hexamer of a bipartite structure composed of a membrane-proximal ATPase domain and a membrane-distal substrate-recognition assembly. The presented information demonstrates the architecture and functional dissection of the multiprotein T4CP complexes and provides important insights into their substrate recruitment and processing.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Legionella pneumophila/química , Complejos Multiproteicos/química , Sistemas de Secreción Tipo IV/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Dominios Proteicos , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Robust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.
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Proteínas Reguladoras de la Apoptosis/inmunología , ADN/inmunología , Herpesvirus Humano 1/inmunología , Inmunidad Innata , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Proteínas Relacionadas con la Autofagia , Beclina-1 , Línea Celular , ADN/metabolismo , Humanos , Interferones/inmunología , Interferones/metabolismo , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismoRESUMEN
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB-Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.