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1.
Nat Immunol ; 15(11): 1079-89, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25282160

RESUMEN

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.


Asunto(s)
Caspasas/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Ribonucleasas/metabolismo , Células Th17/citología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/inmunología , Diferenciación Celular/inmunología , Línea Celular , Genes rel/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Factores Reguladores del Interferón/genética , Interleucina-6/genética , Péptidos y Proteínas de Señalización Intracelular , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas Nucleares/genética , Proteínas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Células Th17/inmunología , Ubiquitina-Proteína Ligasas/genética
2.
Proc Natl Acad Sci U S A ; 120(48): e2309205120, 2023 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-37988467

RESUMEN

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.


Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental , Ratones , Animales , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Inflamación/metabolismo , Diferenciación Celular , Encefalomielitis Autoinmune Experimental/genética , Receptores de Antígenos de Linfocitos T/genética , Ubiquitina-Proteína Ligasas
3.
Nature ; 567(7746): 113-117, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30787442

RESUMEN

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.


Asunto(s)
Centrosoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Microtúbulos/metabolismo , Neurogénesis , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Uniones Intercelulares/metabolismo , Interfase , Ventrículos Laterales/anatomía & histología , Glándulas Mamarias Animales/citología , Ratones , Tamaño de los Órganos , Organoides/citología
4.
EMBO J ; 39(16): e103373, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32627867

RESUMEN

TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular , Proteínas de Unión al ADN/metabolismo , Células-Madre Neurales/metabolismo , Membrana Nuclear/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Ratones , Membrana Nuclear/genética , Dominios Proteicos
5.
Int J Mol Sci ; 25(17)2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39273216

RESUMEN

Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rß, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1-/-/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rß and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rß and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1-/-/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rß in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1-/-/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells-an effect that is most likely facilitated via a decreased expression of PDGF-Rß.


Asunto(s)
Becaplermina , Proliferación Celular , Galectina 1 , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Humanos , Galectina 1/metabolismo , Galectina 1/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Becaplermina/metabolismo , Becaplermina/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Línea Celular , Células Epiteliales/metabolismo
6.
J Neurosci ; 42(8): 1557-1573, 2022 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-34965974

RESUMEN

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENT Dystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.


Asunto(s)
Cannabinoides , Colágeno Tipo VI , Distonía , Trastornos Distónicos , Neuronas Motoras , Plasticidad Neuronal , Animales , Cannabinoides/metabolismo , Cannabinoides/farmacología , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Distonía/genética , Distonía/metabolismo , Trastornos Distónicos/genética , Trastornos Distónicos/metabolismo , Femenino , Masculino , Ratones , Neuronas Motoras/efectos de los fármacos , Mutación , Plasticidad Neuronal/efectos de los fármacos , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/metabolismo
7.
EMBO Rep ; 22(3): e51009, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33512761

RESUMEN

Histone post-translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co-activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ.


Asunto(s)
Histonas , Nucleosomas , Cromatina/genética , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Procesamiento Proteico-Postraduccional
8.
EMBO Rep ; 22(6): e49568, 2021 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-33969602

RESUMEN

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.


Asunto(s)
ADN Circular , Virus de la Hepatitis B , Antivirales/farmacología , Citidina Desaminasa , ADN Circular/genética , ADN Viral/genética , ADN Viral/farmacología , Exorribonucleasas , Virus de la Hepatitis B/genética , Humanos , Interferones , Proteínas , Replicación Viral
9.
Int J Mol Sci ; 24(16)2023 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-37628816

RESUMEN

In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1-/-) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1-/- cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1-/- cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1-/- cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.


Asunto(s)
Galectina 1 , ARN Guía de Sistemas CRISPR-Cas , Humanos , Galectina 1/genética , Actinas , Células Epiteliales , Pigmentos Retinianos
10.
Immunity ; 38(4): 655-68, 2013 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-23583643

RESUMEN

The Roquin-1 protein binds to messenger RNAs (mRNAs) and regulates gene expression posttranscriptionally. A single point mutation in Roquin-1, but not gene ablation, increases follicular helper T (Tfh) cell numbers and causes lupus-like autoimmune disease in mice. In T cells, we did not identify a unique role for the much lower expressed paralog Roquin-2. However, combined ablation of both genes induced accumulation of T cells with an effector and follicular helper phenotype. We showed that Roquin-1 and Roquin-2 proteins redundantly repressed the mRNA of inducible costimulator (Icos) and identified the Ox40 costimulatory receptor as another shared mRNA target. Combined acute deletion increased Ox40 signaling, as well as Irf4 expression, and imposed Tfh differentiation on CD4(+) T cells. These data imply that both proteins maintain tolerance by preventing inappropriate T cell activation and Tfh cell differentiation, and that Roquin-2 compensates in the absence of Roquin-1, but not in the presence of its mutated form.


Asunto(s)
Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , ARN Mensajero/metabolismo , Receptores OX40/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antígenos CD4/metabolismo , Diferenciación Celular/genética , Células HEK293 , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Unión Proteica , Receptores OX40/genética , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genética
11.
Nature ; 515(7526): 287-90, 2014 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-25209665

RESUMEN

Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit. Here we report the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl-Unr-msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. Our results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión , Línea Celular , Respuesta al Choque por Frío , Cristalografía por Rayos X , Compensación de Dosificación (Genética) , Drosophila melanogaster/genética , Femenino , Regulación de la Expresión Génica , Masculino , Modelos Moleculares , Difracción de Neutrones , Resonancia Magnética Nuclear Biomolecular , Motivos de Nucleótidos , Estructura Terciaria de Proteína , ARN Mensajero/química , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Difracción de Rayos X
12.
Mol Cell Proteomics ; 16(8): 1528-1546, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28576849

RESUMEN

Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here, we aim to identify novel interacting binding partners for the ß-galactoside-binding proteins galectin-1 (Gal-1) and galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion, and epithelial-to-mesenchymal transition of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unraveling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pulldown experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor ß PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced cross-linking with LRP1/PDGFRB and integrin-ß1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with kifunensine, an inhibitor of complex-type N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.


Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Galectina 1/metabolismo , Galectina 3/metabolismo , Proteoma/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alcaloides/farmacología , Proteínas Sanguíneas , Membrana Celular/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Inhibidores Enzimáticos/farmacología , Galectinas , Glicosilación/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo
13.
Nucleic Acids Res ; 45(21): 12536-12550, 2017 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-29040648

RESUMEN

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.


Asunto(s)
Proteínas de Drosophila/metabolismo , ARN Helicasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Animales , Proteínas Argonautas/metabolismo , Células Cultivadas , Drosophila/metabolismo , Unión Proteica , Dominios Proteicos , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/química , Proteínas de Unión al ARN/química , Termodinámica
14.
J Struct Biol ; 203(2): 71-80, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29545204

RESUMEN

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.


Asunto(s)
Baculoviridae/genética , Vectores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animales , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Células Sf9
15.
Proc Natl Acad Sci U S A ; 111(1): E34-43, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24351927

RESUMEN

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site ((1441)RASpS(1444)). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis.


Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Mutación , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Alanina/química , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie
16.
Angew Chem Int Ed Engl ; 56(32): 9322-9325, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28636238

RESUMEN

Multi-domain proteins play critical roles in fine-tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi-domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA-1 using Sortase A mediated protein ligation. We show that domain-selective perdeuteration combined with contrast-matched small-angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi-domain proteins and changes induced by ligand binding.


Asunto(s)
Proteínas con Motivos de Reconocimiento de ARN/química , Humanos , Difracción de Neutrones , Conformación Proteica , Dispersión del Ángulo Pequeño
17.
J Virol ; 89(18): 9281-7, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26136564

RESUMEN

UNLABELLED: The hepatitis B virus (HBV) particle is an icosahedral nucleocapsid surrounded by a lipid envelope containing viral surface proteins. A small domain (matrix domain [MD]) in the large surface protein L and a narrow region (matrix binding domain [MBD]) including isoleucine 126 on the capsid surface have been mapped, in which point mutations such as core I126A specifically blocked nucleocapsid envelopment. It is possible that the two domains interact with each other during virion morphogenesis. By the systematic evolution of ligands by exponential enrichment (SELEX) method, we evolved DNA aptamers from an oligonucleotide library binding to purified recombinant capsids but not binding to the corresponding I126A mutant capsids. Aptamers bound to capsids were separated from unbound molecules by filtration. After 13 rounds of selections and amplifications, 16 different aptamers were found among 73 clones. The four most frequent aptamers represented more than 50% of the clones. The main aptamer, AO-01 (13 clones, 18%), showed the lowest dissociation constant (Kd) of 180 ± 82 nM for capsid binding among the four molecules. Its Kd for I126A capsids was 1,306 ± 503 nM. Cotransfection of Huh7 cells with AO-01 and an HBV genomic construct resulted in 47% inhibition of virion production at 3 days posttransfection, but there was no inhibition by cotransfection of an aptamer with a random sequence. The half-life of AO-01 in cells was 2 h, which might explain the incomplete inhibition. The results support the importance of the MBD for nucleocapsid envelopment. Inhibiting the MD-MBD interaction with a low-molecular-weight substance might represent a new approach for an antiviral therapy. IMPORTANCE: Approximately 240 million people are persistently infected with HBV. To date, antiviral therapies depend on a single target, the viral reverse transcriptase. Future additional targets could be viral protein-protein interactions. We selected a 55-base-long single-stranded DNA molecule (aptamer) which binds with relatively high affinity to a region on the HBV capsid interacting with viral envelope proteins during budding. This aptamer inhibits virion formation in cell culture. The results substantiate the current model for HBV morphogenesis and show that the capsid envelope interaction is a potential antiviral target.


Asunto(s)
Antivirales , Aptámeros de Nucleótidos , Cápside/metabolismo , Virus de la Hepatitis B/fisiología , Ensamble de Virus/efectos de los fármacos , Sustitución de Aminoácidos , Antivirales/química , Antivirales/farmacología , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Línea Celular Tumoral , Humanos , Mutación Missense , Estructura Terciaria de Proteína , Ensamble de Virus/genética
18.
J Exp Bot ; 66(3): 989-99, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25428993

RESUMEN

Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that-amongst the 10 Tyr residues present in MSD1-Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.


Asunto(s)
Arabidopsis/genética , Ácido Peroxinitroso/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Superóxido Dismutasa/genética , Tirosina/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Plantas/química , Alineación de Secuencia , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo
19.
Anal Bioanal Chem ; 406(22): 5507-12, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24928115

RESUMEN

In human medicine, procalcitonin (PCT) is a very common and well-established biomarker for sepsis. Even though sepsis is also a leading cause of death in foals and adult horses, up to now, no data about the role of equine PCT in septic horses has been available. Based on monoclonal antibodies targeted against human PCT, we report here the development of a sandwich ELISA for the quantification of equine PCT in equine plasma samples. The ELISA was characterized for intra- and interassay variance and a working range from 25 to 1,000 ng mL(-1) was defined as within this range; both intra- and interassay variances were below 15 %. The target recovery ranged between 73 and 106 %. The ELISA was used to determine the equine PCT concentration in 24 healthy and 5 septic horses to show the potential for clinical evaluation of equine PCT. Significantly different (P = 0.0006) mean equine PCT concentrations were found for the healthy control group and the sepsis group (47 and 8,450 ng mL(-1)).


Asunto(s)
Biomarcadores/sangre , Calcitonina/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Precursores de Proteínas/sangre , Sepsis/diagnóstico , Animales , Anticuerpos Monoclonales/química , Péptido Relacionado con Gen de Calcitonina , Femenino , Caballos , Humanos , Inflamación , Masculino , Curva ROC , Proteínas Recombinantes/química , Reproducibilidad de los Resultados , Sepsis/sangre
20.
Proc Natl Acad Sci U S A ; 108(9): 3554-9, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21321225

RESUMEN

In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific single-substrate enzymes (HisA and TrpF), sharing a similar (ß/α)(8)-barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme.


Asunto(s)
Proteínas Bacterianas/metabolismo , Dominio Catalítico , Histidina/biosíntesis , Isomerasas/química , Isomerasas/metabolismo , Mycobacterium tuberculosis/enzimología , Triptófano/biosíntesis , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Biocatálisis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Isomerasas/antagonistas & inhibidores , Isomerismo , Ligandos , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Estructura Secundaria de Proteína , Especificidad por Sustrato/efectos de los fármacos
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