RESUMEN
Chlordecone (CLD) is a pesticide persisting in soils and contaminating food webs. CLD is sequestered in the liver and poorly metabolized into chlordecol (CLDOH). In vitro liver cell models were used to investigate the fate and mechanistic effects of CLD and CLDOH using multiomics. A 3D-cell model was used to investigate whether CLD and CLDOH can affect susceptibility to the metabolic dysfunction-associated steatotic liver disease (MASLD). Hepatocytes were more sensitive to CLD than CLDOH. CLDOH was intensively metabolized into a glucuronide conjugate, whereas CLD was sequestered. CLD but not CLDOH induced a depletion of Septin-2,- 7,- 9,- 10,- 11 due to proteasomal degradation. Septin binding with CLD and CLDOH was confirmed by surface plasmon resonance. CLD disrupted lipid droplet size and increased saturated long-chain dicarboxylic acid production by inhibiting stearoyl-CoA desaturase (SCD) abundance. Neither CLD nor CLDOH induced steatosis, but CLD induced fibrosis in the 3D model of MASLD. To conclude, CLD hepatoxicity is specifically driven by the degradation of septins. CLDOH, was too rapidly metabolized to induce septin degradation. We show that the conversion of CLD to CLDOH reduced hepatotoxicity and fibrosis in liver organoids. This suggests that protective strategies could be explored to reduce the hepatotoxicity of CLD.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatocitos , Complejo de la Endopetidasa Proteasomal , Septinas , Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Septinas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Insecticidas/toxicidad , Insecticidas/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/inducido químicamente , Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/patologíaRESUMEN
Collagen Q (ColQ) is a nonfibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on muscle-specific kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with low-density lipoprotein receptor-related protein 4 (LRP4), its coreceptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK-LRP4 activation. Using coimmunoprecipitation, pull-down, plate-binding assays, and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show that the LRP4 N-terminal region, which contains the agrin-binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK-LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes but concomitantly increases MuSK accumulation at the muscle cell surface. Our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and acetylcholinesterase anchoring at the NMJ.
Asunto(s)
Acetilcolinesterasa , Agrina , Colágeno , Unión Neuromuscular , Humanos , Acetilcolinesterasa/metabolismo , Agrina/genética , Agrina/metabolismo , Colágeno/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Unión Neuromuscular/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Monocytes are considered crucial actors of inflammation in sickle cell disease (SCD), being responsible for an increased production of proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6. Although a role of free heme released by intravascular hemolysis has been suspected, the mechanisms underlying monocyte activation in patients with SCD remain unknown. Using purified human hemoglobin (Hb), we demonstrate herein, that cell-free HbS, unlike HbA or heme, is responsible for a major enhancement in the expression of proinflammatory cytokines by human monocytes. This effect was found mediated by direct interaction with the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex, resulting in the activation of both the nuclear factor-κB (NF-κB) and type I interferon pathways. In Townes SCD mice, injection of HbS, unlike HbA, was responsible for an increased production of proinflammatory cytokines, which was prevented by the TLR4 inhibitor, TAK-242. Our results reveal a novel mechanism of monocyte activation and systemic inflammation in SCD, which opens new promising therapeutic perspectives targeting the HbS-TLR4 interaction.
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Anemia de Células Falciformes , Receptor Toll-Like 4 , Humanos , Ratones , Animales , Receptor Toll-Like 4/metabolismo , Monocitos/metabolismo , Transducción de Señal , FN-kappa B/metabolismo , Citocinas/metabolismo , Inflamación/metabolismo , Anemia de Células Falciformes/metabolismo , Hemo/metabolismoRESUMEN
The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunoglobulina A/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Seronegatividad para VIH/inmunología , Humanos , Inmunoglobulina A/química , Inmunoglobulina G/inmunología , Dominios Proteicos/inmunologíaRESUMEN
The authors wish to make the following correction to their published paper [...].
RESUMEN
Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.
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Arabidopsis/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Ácido Salicílico/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cloroplastos/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , NAD , Mutación PuntualRESUMEN
Hybrid nanostructures are constructed by the direct coupling of fluorescent quantum dots and plasmonic gold nanoparticles. Self-assembly is directed by the strong affinity between two artificial α-repeat proteins that are introduced in the capping layers of the nanoparticles at a controlled surface density. The proteins have been engineered to exhibit a high mutual affinity, corresponding to a dissociation constant in the nanomolar range, towards the protein-functionalized quantum dots and gold nanoparticles. Protein-mediated self-assembly is evidenced by surface plasmon resonance and gel electrophoresis. The size and the structure of colloidal superstructures of complementary nanoparticles are analyzed by transmission electron microscopy and small angle X-ray scattering. The size of the superstructures is determined by the number of proteins per nanoparticle. The well-defined geometry of the rigid protein complex sets a highly uniform interparticle distance of 8 nm that affects the emission properties of the quantum dots in the hybrid ensembles. Our results open the route to the design of hybrid emitter-plasmon colloidal assemblies with controlled near-field coupling and better optical response.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Proteínas/química , Puntos Cuánticos/química , Resonancia por Plasmón de Superficie , ElectroforesisRESUMEN
The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Mapeo Epitopo/métodos , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Bacteriófagos/inmunología , VIH-1 , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Simulación del Acoplamiento Molecular , Pruebas de Neutralización , Péptidos/administración & dosificación , Péptidos/inmunología , Unión Proteica/efectos de los fármacosRESUMEN
BACKGROUND: We have previously demonstrated that the eukaryote-specific ribosomal protein eL42 of the human 80S ribosome contains seven monomethylated residues, among which are the Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence conserved in all eukaryotic 80S ribosomes. This sequence contains the methylated and universally conserved GGQ motif common for all class-1 translation termination factors responsible for stop codon recognition and for triggering the hydrolysis of the P site-bound peptidyl-tRNA. We have also recently reported a model of ribosomal ternary eL42-tRNA-eRF1 complex where specific regions of all three macromolecules (the comparably flexible GGQ domains of eRF1 and eL42 and the CCA-arm of tRNA) are involved in interactions. METHOD: Here, we have studied the interactions between recombinant eL42 and eRF1 proteins and the tRNA substrate by means of the Biacore assay, using the wild-type eL42 protein, the eL42-Δ(GGQTK) mutant (the eL42 protein whose GGQTK motif has been deleted), the single Q51E and K53Q mutants (eL42-Q51E and eL42-K53Q, respectively), as well as the double Q51A/K53A mutant (eL42-Q51A/K53A). RESULTS: Our results show that the monomethylated Gln-51 and Lys-53 residues contained in the 47GFGGQTK53 sequence of eL42 and the monomethylated GGQ motif of eRF1 represents the sites of interaction between these two proteins through hydrophobic contacts between methyl groups. We also demonstrate that the interactions between eL42 and tRNA or 28S rRNA are characterized by strong binding affinities (KD values in the nanomolar or picomolar range, respectively) which argue for specific interactions. Strong interactions between eL42 and tRNA are likely to be responsible for the decrease in the poly(U)-dependent poly(Phe) synthesis activity of human 80S or E. coli 70S ribosomes in the presence of added human recombinant eL42. It is proposed that the decrease of the activity of the ribosome is caused by the sequestration of the substrate Phe-tRNAPhe by the added eL42 protein. CONCLUSION: Interactions between the monomethylated Gln-51 and Lys-53 residues of the 49GGQTK53 motif of the human eL42 protein and the methylated GGQ motif of eRF1 are likely to play a functional role on translating human 80S ribosomes.
RESUMEN
Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. STATEMENT OF SIGNIFICANCE: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects.
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Materiales Biomiméticos , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 6 , Proteína Morfogenética Ósea 7 , Materiales Biocompatibles Revestidos , Durapatita , Fibronectinas , Osteoblastos/metabolismo , Titanio , Animales , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 6/química , Proteína Morfogenética Ósea 6/farmacología , Proteína Morfogenética Ósea 7/química , Proteína Morfogenética Ósea 7/farmacología , Línea Celular , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Durapatita/química , Durapatita/farmacología , Fibronectinas/química , Fibronectinas/farmacología , Humanos , Ratones , Osteoblastos/citología , Titanio/química , Titanio/farmacologíaRESUMEN
Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Antiprotozoarios/farmacología , Ampicilina/farmacología , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/toxicidad , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacocinética , Péptidos Catiónicos Antimicrobianos/toxicidad , Antiprotozoarios/química , Antiprotozoarios/farmacocinética , Antiprotozoarios/toxicidad , Apoptosis/efectos de los fármacos , Bacterias/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana , Humanos , Leishmania/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Factores de Tiempo , Trypanosoma/efectos de los fármacos , Liposomas Unilamelares/químicaRESUMEN
The eukaryotic release factor 3 (eRF3) has been involved in the control of mRNA degradation through its association with the cytoplasmic Poly(A) Binding Protein, PABP. In mammals, eRF3 N-terminal domain contains two overlapping PAM2 motifs which specifically recognize the MLLE domain of PABP. In humans, eRF3a/GSPT1 gene contains a stable GGC repeat encoding a repeat of glycine residues in eRF3a N-terminus. There are five known eRF3a/GSPT1 alleles in the human population, encoding 7, 9, 10, 11 and 12 glycines. Several studies have reported that the presence of eRF3a 12-GGC allele is correlated with an increased risk of cancer development. Using surface plasmon resonance, we have studied the interaction of the various allelic forms of eRF3a with PABP alone or poly(A)-bound PABP. We found that the N-terminal glycine repeat of eRF3a influences eRF3a-PABP interaction and that eRF3a 12-GGC allele has a decreased binding affinity for PABP. Our comparative analysis on eRF3a alleles suggests that the presence of eRF3a 12-GGC allele could modify the coupling between translation termination and mRNA deadenylation.
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Variación Genética , Glicina/genética , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Alelos , Sitios de Unión , Predisposición Genética a la Enfermedad , Humanos , Modelos Moleculares , Neoplasias/genética , Factores de Terminación de Péptidos/química , Unión Proteica , ARN Mensajero/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHß transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.
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Enanismo Hipofisario/genética , Mutación Missense , Carácter Cuantitativo Heredable , Factor de Transcripción Pit-1/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Enanismo Hipofisario/metabolismo , Enanismo Hipofisario/patología , Femenino , Regulación de la Expresión Génica , Genes Dominantes , Sitios Genéticos , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Heterocigoto , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Linaje , Hipófisis/metabolismo , Hipófisis/patología , Prolactina/genética , Prolactina/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factor de Transcripción Pit-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , ADN Bacteriano/genética , ADN Complementario/genética , Placozoa/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Candida/efectos de los fármacos , Línea Celular Tumoral , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Cinética , Membranas/efectos de los fármacos , Modelos Biológicos , Datos de Secuencia Molecular , Placozoa/genética , Estructura Secundaria de Proteína , Ratas , Alineación de Secuencia , Resonancia por Plasmón de Superficie , Células U937RESUMEN
Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.
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Quinasa 1 de Adhesión Focal/química , Secuencias de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Activación Enzimática , Quinasa 1 de Adhesión Focal/fisiología , Adhesiones Focales , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Fosfotirosina/fisiología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Dispersión de RadiaciónRESUMEN
Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT(1A)R in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons.
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Dendritas/fisiología , Regiones de Fijación a la Matriz/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Receptor de Serotonina 5-HT1A/fisiología , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/fisiología , Animales , Células Cultivadas , Dendritas/genética , Marcación de Gen/métodos , Humanos , Regiones de Fijación a la Matriz/genética , Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1A/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Corto's chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA-seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Complejo Represivo Polycomb 1/metabolismo , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Expresión Génica , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas HSP70 de Choque Térmico/genética , Lisina/metabolismo , Metilación , Datos de Secuencia Molecular , Fenotipo , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Alineación de Secuencia , Transcripción Genética , TranscriptomaRESUMEN
The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Inmunoglobulina G/inmunología , Síndrome de Inmunodeficiencia Adquirida/terapia , Síndrome de Inmunodeficiencia Adquirida/transmisión , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Epítopos/genética , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/farmacología , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/patogenicidad , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Estructura Secundaria de Proteína , Transcitosis/efectos de los fármacos , Transcitosis/inmunologíaRESUMEN
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A(2)/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44-77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120-210). A 25-mer peptide named P46 (aa 46-70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.