RESUMEN
Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus, the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.
Asunto(s)
Modelos Moleculares , Conformación Proteica , ARN Polimerasa Dependiente del ARN/química , Virus de la Fiebre del Valle del Rift/enzimología , Secuencias de Aminoácidos , Dominio Catalítico , Fenómenos Químicos , Secuencia Conservada , Microscopía por Crioelectrón , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Virales/químicaRESUMEN
Curli play critical roles in biofilm formation, host cell adhesion, and colonization of inert surfaces in many Enterobacteriaceae. In Escherichia coli, curli biogenesis requires 7 curli-specific gene (csg) products-CsgA through G-working in concert. Of them, CsgG and CsgF are 2 outer membrane (OM)-localized components that consists of the core apparatus for secretion and assembly of curli structural subunits, CsgB and CsgA. Here, we report the cryogenic electron microscopy (cryo-EM) structure of CsgG in complex with CsgF from E. coli. The structure reveals that CsgF forms a stable complex with CsgG via a 1:1 stoichiometry by lining the upper lumen of the nonameric CsgG channel via its N-terminal 27 residues, forming a funnel-like entity plugged in the CsgG channel and creating a unique secretion channel with 2 constriction regions, consistent with the recently reported structure of the CsgG-CsgF complex. Functional studies indicate that export of CsgF to the cell surface requires the CsgG channel, and CsgF not only functions as an adaptor that bridges CsgB with CsgG but also may play important roles in controlling the rates of translocation and/or polymerization for curli structural subunits. Importantly, we found that a series of CsgF-derived peptides are able to efficiently inhibit curli production to E. coli when administrated exogenously, highlighting a potential strategy to interfere biofilm formation in E. coli strains.
Asunto(s)
Amiloide/ultraestructura , Microscopía por Crioelectrón , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Complejos Multiproteicos/ultraestructura , Multimerización de Proteína , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Unión ProteicaRESUMEN
ß-barrel outer membrane proteins (ß-OMPs) play critical roles in nutrition acquisition, protein import/export, and other fundamental biological processes. The assembly of ß-OMPs in Gram-negative bacteria is mediated by the ß-barrel assembly machinery (BAM) complex, yet its precise mechanism remains elusive. Here, we report two structures of the BAM complex in detergents and in nanodisks, and two crystal structures of the BAM complex with bound substrates. Structural analysis indicates that the membrane compositions surrounding the BAM complex could modulate its overall conformations, indicating low energy barriers between different conformational states and a highly dynamic nature of the BAM complex. Importantly, structures of the BAM complex with bound substrates and the related functional analysis show that the first ß-strand of the BamA ß-barrel (ß1BamA ) in the BAM complex is associated with the last but not the first ß-strand of a ß-OMP substrate via antiparallel ß-strand interactions. These observations are consistent with the ß-signal hypothesis during ß-OMP biogenesis, and suggest that the ß1BamA strand in the BAM complex may interact with the last ß-strand of an incoming ß-OMP substrate upon their release from the chaperone-bound state.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Aprendizaje Automático , Conformación Proteica en Lámina beta , Dominios ProteicosRESUMEN
Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and glycine, which is crucial for one carbon metabolism. Here, we report the first crystal structure of cytoplasmic SHMT from Pichia pastoris (pcSHMT) diffracted to 2.5â¯Å resolution in space group C2221. PcSHMT was a contaminant with our target protein expressed in Pichia pastoris and confirmed by mass spectrometry. The overall structure of pcSHMT is similar to Human mitochondrial SHMT and different to E. coli SHMT. Interestingly, the oligomerization of pcSHMT expressed in eukaryotic or prokaryotic system differs significantly and is regulated by pyridoxal-5'-phosphate. Our results revealed a close evolutionary relationship between Pichia pastoris and Human mitochondria.
Asunto(s)
Glicina Hidroximetiltransferasa/metabolismo , Pichia/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Glicina Hidroximetiltransferasa/química , Humanos , Modelos Moleculares , Pichia/química , Pichia/citología , Pichia/metabolismo , Conformación Proteica , Multimerización de Proteína , Fosfato de Piridoxal/metabolismoRESUMEN
Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the United States and almost 1.5 billion in Europe every year. To find a way to combat these viruses, we focused on the essential viral nonstructural protein 11 (nsp11). nsp11 is associated with multiple functions, such as RNA processing and suppression of the infected host innate immunity system. The three structures solved in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.
Asunto(s)
Endorribonucleasas/química , Equartevirus/química , Virus del Síndrome Respiratorio y Reproductivo Porcino/química , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Equartevirus/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Modelos Moleculares , Mutación , Virus del Síndrome Respiratorio y Reproductivo Porcino/enzimología , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Alineación de Secuencia , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Forkhead box-O1 (FoxO1) is a key nutrient- and growth factor-dependent regulator of metabolism, but its functional role in human primary keratinocytes (HPKs) is less known. To investigate the role of FoxO1 in HPKs and effect of insulin-like growth factor 1 (IGF-1) and isotretinoin on FoxO1 expression, HPKs were treated with 1.2 mmol/L calcium chloride, 1-20 ng/mL IGF-1 and 0.1-10 µmol/L isotretinoin. Recombinant adenovirus expressing FoxO1 or FKHR shRNA lentivirus transfection was introduced to upregulate or silence FoxO1 expression. Epidermal FoxO1 immunostaining was lower in acne lesion than in normal skin. FoxO1 overexpression induced involucrin expression, G2/M arrest and apoptosis but suppressed proliferation, while FoxO1 silencing decreased involucrin expression but increased proliferation, S phase and viable cells in HPKs. IGF-1 downregulated FoxO1 and involucrin but upregulated p-Akt expression in HPKs, which was blocked by pretreatment with LY294002. Isotretinoin enhanced FoxO1, p53 and p21 but inhibited p-FoxO1 and involucrin expression in HPKs. These results demonstrate that FoxO1 promotes differentiation and apoptosis in HPKs. IGF-1 may reduce keratinocyte differentiation through PI3K/Akt/FoxO1 pathway, while isotretinoin can reinforce FoxO1 expression. FoxO1 may be involved in acne pathogenesis and could serve as a potential therapeutic target.
Asunto(s)
Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Diferenciación Celular/genética , Proteína Forkhead Box O1/genética , Queratinocitos/fisiología , Acné Vulgar/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Cromonas/farmacología , Fármacos Dermatológicos/farmacología , Inhibidores Enzimáticos/farmacología , Proteína Forkhead Box O1/metabolismo , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Isotretinoína/farmacología , Morfolinas/farmacología , Fosforilación , Cultivo Primario de Células , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaF2E2B2D6, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Complejos Multiproteicos/metabolismo , Fosfolípidos/metabolismo , Pseudomonas aeruginosa/metabolismo , Adenosina Difosfato/metabolismo , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Transporte Biológico , Reactivos de Enlaces Cruzados/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Autoimmune progesterone dermatitis (APD) is a rare cutaneous disorder with cyclic skin eruptions during the luteal phase of the menstrual cycle. Patients can present with various clinical manifestations, including urticaria and angioedema, erythema multiforme, eczema, fixed drug eruption and centrifugal erythema annulare. In our case, however, the patient's skin lesions mimic necrotic migratory erythema (NME) which is most commonly associated with glucagonoma and rarely with liver disease, inflammatory bowel disease, malnutrition and other tumors. To our knowledge, this is the first case of NME-like APD and is successfully controlled by danazol. This also sheds lights on the etiologic diversity of NME.
Asunto(s)
Enfermedades Autoinmunes/diagnóstico , Danazol/uso terapéutico , Dermatitis/diagnóstico , Antagonistas de Estrógenos/uso terapéutico , Eritema Necrolítico Migratorio/diagnóstico , Progesterona/efectos adversos , Adulto , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Dermatitis/complicaciones , Dermatitis/tratamiento farmacológico , Dermatitis/inmunología , Diagnóstico Diferencial , Femenino , Glucagonoma/complicaciones , Glucagonoma/diagnóstico , Humanos , Eritema Necrolítico Migratorio/tratamiento farmacológico , Eritema Necrolítico Migratorio/inmunología , Neoplasias Pancreáticas/complicaciones , Neoplasias Pancreáticas/diagnóstico , Progesterona/inmunología , Piel/inmunología , Piel/patología , Pruebas Cutáneas , Resultado del TratamientoRESUMEN
Relative of Early Flowing 6 (REF6) is a DNA-sequence-specific H3K27me3/2 demethylase that contains four zinc finger (ZnF) domains and targets several thousand genes in Arabidopsis thaliana. The ZnF domains are essential for binding target genes, but the structural basis remains unclear. Here, we determined crystal structures of the ZnF domains and REF6-DNA complex, revealing a unique REF6-family-specific half-cross-braced ZnF (RCZ) domain and two C2H2-type ZnFs. DNA-binding induces a profound conformational change in the hinge region of REF6. Each REF6 recognizes six bases and DNA methylation reduces the binding affinity. Both the acidic region and basic region are important for the self-association of REF6. The REF6 DNA-binding affinity is determined by the sequence-dependent conformations of DNA and also the cooperativity in different target motifs. The conformational plasticity enables REF6 to function as a global transcriptional regulator that directly binds to many diverse genes, revealing the structural basis for the epigenetic modification recognition.
RESUMEN
Sebocyte differentiation is a continuous process, but its potential molecular mechanism remains unclear. We aimed to establish a novel sebocyte differentiation model using human primary sebocytes and to identify the expression profiles of differentiation-associated proteins. Primary human sebocytes were cultured on Sebomed medium supplemented with 2% serum for 7 days. Flow cytometry showed that S phase cells were decreased time-dependently, while G1 and subG1 (apoptosis) phase cells increased under serum starvation. Transmission electron microscopy and Oil Red O staining revealed a gradual increase of intracellular lipid accumulation. Expression of proliferation marker was diminished, while expression of differentiation, apoptosis, and lipogenic markers elevated gradually during 7-day culture. iTRAQ analysis identified 3582 expressed proteins in this differentiation model. Compared with day 0, number of differentially expressed proteins was 132, 54, 321, and 96 at days 1, 3, 5, and 7, respectively. Two overexpressed proteins (S100 calcium binding protein P and ferredoxin reductase) and 2 downexpressed proteins (adenosine deaminase and keratin 10) were further confirmed by Western blot and immunohistochemistry.
Asunto(s)
Diferenciación Celular , Células Epiteliales/citología , Modelos Biológicos , Proteoma/metabolismo , Proteómica/métodos , Sebo/citología , Acné Vulgar/patología , Apoptosis , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Lipogénesis , Reproducibilidad de los Resultados , Piel/patologíaRESUMEN
BACKGROUND: The transcription factor Sox9 is pivotal in the morphogenesis of hair follicles, but its role in sebocytes is poorly understood. OBJECTIVE: We investigated the effect of Sox9 on human sebocyte proliferation, differentiation and lipogenesis. METHODS: Sox9 expression was detected by immunohistochemistry in normal skin and acne lesion. Primary cultured human sebocytes were transfected with adenovirus expressing GFP-Sox9 or Sox9 microRNA. Sox9 and peroxisome proliferator-activated receptor (PPAR)γ expression in sebocytes was detected by quantitative real-time PCR, Western blot and immunocytofluorescence; cell proliferation was measured by MTS and [3H]-thymidine incorporation assays; cell cycle distribution and apoptosis were evaluated by propidium iodide staining-based flow cytometry; and intracellular lipid levels were assessed by Oil Red O stain. RESULTS: Sox9 immunostaining was increased in mature sebocytes of acne lesion compared with normal skin. Expression of Sox9 mRNA and protein and PPARγ protein was elevated with cell confluent levels in sebocytes. Sox9 overexpression enhanced proliferation, differentiation, proportion of S and G2/M cells, lipogenesis and PPARγ expression in sebocytes, while Sox9 silencing caused inhibition of differentiation, lipogenesis and PPARγ expression, and increase of G1 and sub-G1 (apoptotic) cell fraction. The suppression of Sox9 knockdown on sebocyte growth was observed using [3H]-thymidine incorporation but not MTS assay. CONCLUSION: These results demonstrate that Sox9 can reinforce sebocyte proliferation, differentiation and lipogenesis. The G1/S transition arrest and apoptotic induction might contribute to inhibitory effect of Sox9 silencing on sebocyte proliferation.
Asunto(s)
Acné Vulgar/metabolismo , Diferenciación Celular , Proliferación Celular , Lipogénesis , Factor de Transcripción SOX9/metabolismo , Glándulas Sebáceas/citología , Acné Vulgar/patología , Apoptosis , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Fase G1 , Humanos , Receptores Activados del Proliferador del Peroxisoma , Cultivo Primario de Células , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción SOX9/genética , Cuero Cabelludo/citologíaRESUMEN
Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.