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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810359

RESUMEN

Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.


Asunto(s)
Vesículas Extracelulares/trasplante , Hipersensibilidad Retardada/terapia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunomodulación , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Linfocitos T/inmunología , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , Citocinas/farmacología , Pulpa Dental/citología , Vesículas Extracelulares/inmunología , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lentivirus/genética , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfocitos T/fisiología , Telomerasa/genética , Telomerasa/metabolismo , Adulto Joven
2.
Nat Commun ; 12(1): 1577, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707427

RESUMEN

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Asunto(s)
/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , /inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , /química , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Afinidad de Anticuerpos , Línea Celular , Chlorocebus aethiops , Biblioteca de Genes , Voluntarios Sanos , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/aislamiento & purificación , Modelos Moleculares , Mutación , Pruebas de Neutralización , Pandemias , Biblioteca de Péptidos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
3.
Sheng Wu Gong Cheng Xue Bao ; 37(3): 939-949, 2021 Mar 25.
Artículo en Chino | MEDLINE | ID: mdl-33783159

RESUMEN

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.


Asunto(s)
6-Fitasa , Pichia , 6-Fitasa/genética , Pichia/genética , Plásmidos , Proteínas Recombinantes/genética , Saccharomycetales
4.
Nat Commun ; 12(1): 1950, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782388

RESUMEN

Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, impacts millions of people. Entry into target cells is mediated by the HIV-1 envelope (Env) glycoprotein interacting with host receptor CD4, which triggers conformational changes allowing binding to a coreceptor and subsequent membrane fusion. Small molecule or peptide CD4-mimetic drugs mimic CD4's Phe43 interaction with Env by inserting into the conserved Phe43 pocket on Env subunit gp120. Here, we present single-particle cryo-EM structures of CD4-mimetics BNM-III-170 and M48U1 bound to a BG505 native-like Env trimer plus the CD4-induced antibody 17b at 3.7 Å and 3.9 Å resolution, respectively. CD4-mimetic-bound BG505 exhibits canonical CD4-induced conformational changes including trimer opening, formation of the 4-stranded gp120 bridging sheet, displacement of the V1V2 loop, and formation of a compact and elongated gp41 HR1C helical bundle. We conclude that CD4-induced structural changes on both gp120 and gp41 Env subunits are induced by binding to the gp120 Phe43 pocket.


Asunto(s)
Antígenos CD4/química , Guanidinas/química , Proteína gp120 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Indenos/química , Receptores Virales/química , Animales , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Antígenos CD4/antagonistas & inhibidores , Antígenos CD4/genética , Antígenos CD4/metabolismo , Células CHO , Clonación Molecular , Cricetulus , Microscopía por Crioelectrón , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Guanidinas/metabolismo , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Indenos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores Virales/antagonistas & inhibidores , Receptores Virales/genética , Receptores Virales/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo
5.
mBio ; 12(2)2021 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-33727353

RESUMEN

The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.


Asunto(s)
/química , Betacoronavirus/fisiología , Replicación Viral , Secuencia de Aminoácidos , Animales , Betacoronavirus/metabolismo , Sitios de Unión , Línea Celular , Infecciones por Coronavirus/virología , Especificidad del Huésped , Humanos , Ratones , Modelos Moleculares , Mutación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , /fisiología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo
6.
Sci Signal ; 14(675)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758061

RESUMEN

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Asunto(s)
/metabolismo , Señalización del Calcio/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , NADP/análogos & derivados , /fisiología , Marcadores de Afinidad , Animales , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Química Clic/métodos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transcriptoma , Internalización del Virus
7.
Nat Commun ; 12(1): 1413, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658493

RESUMEN

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).


Asunto(s)
Exocitosis/fisiología , Proteínas Luminiscentes/metabolismo , Animales , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/genética , Mutación , Neuronas/citología , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/fisiología , Imagen de Lapso de Tiempo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
8.
Biomed Khim ; 67(1): 66-73, 2021 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-33645523

RESUMEN

The homodimeric glycoprotein, anti-mullerian hormone (AMH), described over 70 years ago by A. Jost, is the least studied member of the transforming growth factor beta superfamily. Despite the antitumor activity of AMH discovered at the end of the last century, the creation of effective drugs based on AMH is hindered primarily by the lack of information on the mechanism of various AMH forms interaction with a specific type II receptor (MISRII). Previously, we have shown that not only the full-length activated hormone but also its C-terminal fragment (C-rAMH) could bind to MISRII. In this work, using the surface plasmon resonance technique, we compared the interaction of three forms of recombinant AMH (rAMH) with the MISRII analogue - the chimeric protein MISRII-Fc containing AMH type II receptor and a Fc-fragment of the human IgG1 heavy chain. Comparison of the binding of MISRII-Fc, immobilized on a chip with group specificity for human immunoglobulins, to C-rAMH, to intact rAMH (pro-rAMH), and to rAMH containing one uncleaved monomer (hc-rAMH), showed that the KD of the complexes increased: 1.7 nM, 88 nM and 110 nM, respectively. Thus, we have shown that C-terminal fragment of AMH has the maximum affinity for the recombinant MISRII analogue, which indicates the prospects for the development of drugs based on this hormone derivative.


Asunto(s)
Hormona Antimülleriana , Factor de Crecimiento Transformador beta , Hormona Antimülleriana/genética , Humanos , Proteínas Recombinantes/genética
9.
Food Chem ; 350: 129212, 2021 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-33609939

RESUMEN

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.


Asunto(s)
Aspergillus oryzae/enzimología , Frío , Ingeniería Genética/métodos , Chaperonas Moleculares/genética , Petróleo/análisis , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/metabolismo , Clonación Molecular , Expresión Génica , Concentración de Iones de Hidrógeno , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipasas de Tipo C/genética
10.
Viruses ; 13(2)2021 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-33562288

RESUMEN

Carassius auratus herpesvirus (CaHV) has been identified as a high-virulence pathogenic virus that infects aquatic animals, but the key factor for virus-host interaction is still unclear. Five Really interesting new genes (RING) finger proteins (39L, 52L, 131R, 136L, and 143R) of CaHV were screened to determine structural diversity. RING finger proteins were also predicted in other known fish herpesviruses, with an arrangement and number similar to CaHV. We performed multifaceted analyses of the proteins, including protein sizes, skeleton structures, subcellular localizations, and ubiquitination activities, to determine their precise roles in virus-host interactions. The five proteins were overexpressed and detected different levels of ubiquitination activities, and 143R showed the highest activity. Then, the prokaryotic expressed and purified full-length proteins (131R and 136L), RING domain isolates (131R12-43 and 136L45-87), and RING domain-deleted mutants (131RΔ12-43 and 136LΔ45-87) were prepared to detect their activities through ubiquitination assays. The results indicate that both full-length proteins and their isolates have activities that catalyze ubiquitination, and the full-length proteins possess higher activity than the isolates, but RING domain-deleted mutants lose their activities. Furthermore, the activities of the five proteins were verified as E3 ubiquitin ligase activity, showing that the RING domains determine the ubiquitination activity. These proteins present different subcellular localization. RING domain-deleted mutants showed similar subcellular localization with their full-length proteins, and all the isolates diffused in whole cells. The current results indicate that the sequence outside the RING domain determines subcellular localization and the level of ubiquitination activity, suggesting that the RING finger proteins of fish herpesviruses might have diverse functions in virus-host interaction.


Asunto(s)
Herpesviridae/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Enfermedades de los Peces/virología , Carpa Dorada/virología , Células HEK293 , Herpesviridae/genética , Herpesviridae/fisiología , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Interacciones Huésped-Patógeno , Humanos , Espacio Intracelular/metabolismo , Mutación , Dominios RING Finger/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Virales/genética
11.
Arch Virol ; 166(4): 1113-1124, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33576898

RESUMEN

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.


Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Bronquitis Infecciosa/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Enfermedades de las Aves de Corral/diagnóstico , Análisis por Matrices de Proteínas/veterinaria , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Pollos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Inmunoensayo/normas , Inmunoensayo/veterinaria , Virus de la Bronquitis Infecciosa/inmunología , Virus de la Influenza A/inmunología , Gripe Aviar/diagnóstico , Enfermedad de Newcastle/diagnóstico , Virus de la Enfermedad de Newcastle/inmunología , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Pruebas Serológicas/normas , Pruebas Serológicas/veterinaria
12.
Nat Commun ; 12(1): 819, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547302

RESUMEN

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Dominio Catalítico , Clonación Molecular , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Muerte Celular Regulada/genética , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
13.
Nat Commun ; 12(1): 815, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547286

RESUMEN

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.


Asunto(s)
Aminopiridinas/química , Azepinas/química , Antagonistas de los Receptores de Orexina/química , Receptores de Orexina/química , Péptidos/química , Fármacos Inductores del Sueño/química , Sulfonamidas/química , Triazoles/química , Aminopiridinas/metabolismo , Azepinas/metabolismo , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Antagonistas de los Receptores de Orexina/metabolismo , Receptores de Orexina/agonistas , Receptores de Orexina/metabolismo , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fármacos Inductores del Sueño/metabolismo , Sulfonamidas/metabolismo , Triazoles/metabolismo
14.
Nat Commun ; 12(1): 828, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547294

RESUMEN

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.


Asunto(s)
Adenilil Imidodifosfato/química , Proteínas HSP90 de Choque Térmico/química , Chaperonas Moleculares/química , Prostaglandina-E Sintasas/química , Receptores de Glucocorticoides/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenilil Imidodifosfato/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulación de Dinámica Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Prostaglandina-E Sintasas/genética , Prostaglandina-E Sintasas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
15.
Nat Commun ; 12(1): 807, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547325

RESUMEN

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Asunto(s)
Apoproteínas/química , Calcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Sustitución de Aminoácidos , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopía por Crioelectrón , Cisteína/química , Cisteína/metabolismo , Expresión Génica , Humanos , Transporte Iónico , Hipertermia Maligna/genética , Hipertermia Maligna/patología , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Porcinos
16.
Nat Commun ; 12(1): 795, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542217

RESUMEN

Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed digital affinity profiling via proximity ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms are identified. We further demonstrate that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications show a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggests that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.


Asunto(s)
5-Metilcitosina/análogos & derivados , ADN/metabolismo , Epigenómica/métodos , 5-Metilcitosina/metabolismo , Línea Celular , Islas de CpG/genética , ADN/genética , Elementos de Facilitación Genéticos , Epigénesis Genética , Biblioteca de Genes , Células Madre Embrionarias Humanas , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factores Estimuladores hacia 5'/genética , Factores Estimuladores hacia 5'/aislamiento & purificación , Factores Estimuladores hacia 5'/metabolismo
17.
Nat Commun ; 12(1): 1028, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589610

RESUMEN

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.


Asunto(s)
Proteínas Portadoras/química , ADN/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribosa) Polimerasa-1/química , Serina/metabolismo , ADP-Ribosilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática
18.
Virology ; 557: 15-22, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33582454

RESUMEN

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.


Asunto(s)
Anticuerpos Antivirales/sangre , /inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , /inmunología , Secuencia de Aminoácidos , /inmunología , Estudios de Casos y Controles , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Sueros Inmunes/química , Inmunoglobulina M/sangre , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
19.
Nat Commun ; 12(1): 723, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33526787

RESUMEN

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3.


Asunto(s)
Adenocarcinoma/secundario , Neoplasias Óseas/secundario , Células Madre Mesenquimatosas/patología , Neoplasias de la Próstata/patología , Receptores de Interferón/metabolismo , Ácidos Aminosalicílicos/farmacología , Ácidos Aminosalicílicos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Bencenosulfonatos/farmacología , Bencenosulfonatos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo Condicionados/metabolismo , Modelos Animales de Enfermedad , Docetaxel/farmacología , Docetaxel/uso terapéutico , Humanos , Interferones/genética , Interferones/metabolismo , Masculino , Ratones Noqueados , Osteoblastos/patología , Cultivo Primario de Células , Neoplasias de la Próstata/tratamiento farmacológico , ARN Interferente Pequeño/metabolismo , Receptores de Interferón/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Tibia/patología
20.
Nat Commun ; 12(1): 790, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542233

RESUMEN

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flavonas/metabolismo , Flavonoles/metabolismo , Microbioma Gastrointestinal/fisiología , Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Clostridiales/enzimología , Clostridiales/genética , Cristalografía por Rayos X , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/ultraestructura , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura
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