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1.
Small ; : e2310913, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38726952

RESUMEN

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

2.
NPJ Antimicrob Resist ; 1(1): 8, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38686212

RESUMEN

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

3.
Biochim Biophys Acta Biomembr ; 1863(5): 183571, 2021 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-33561475

RESUMEN

Dipalmitoyl-3-aza-dehydroxy-lysylphosphatidylglycerol (DP3adLPG), is a chemically stable synthetic analogue of the bacterial lipid lysylphosphatidylglycerol (LPG), designed as a substitute for the notoriously labile native lipid in biophysical investigations. In Staphylococcus aureus, LPG is known to play a role in resistance to antibiotics by altering membrane charge properties in response to environmental stress, but little is known about how LPG influences other bilayer physicochemical properties or lateral organisation, through the formation of complexes with lipids such as phosphatidylglycerol (PG). In this study we have investigated the different phases formed by biomimetic mixtures of 3adLPG and PG in different thermotropic states, using neutron diffraction and electron microscopy. In a DPPG/DP3adLPG 70:30 mol% mixture, two distinct lamellar phases were observed below the lipid melting transition: Lß' 1 and Lß' 2 with respective periodicities of 82 and 62 Å. Increasing the proportion of DP3adLPG to mimic the effects of environmental stress led to the disappearance of the Lß' 1 phase and the formation of an inverse hexagonal phase. The compositions of these different phases were identified by investigating the thermotropic properties of the two mixtures, and probing their interaction with the antimicrobial peptide magainin 2 F5W. We propose that the observed polymorphism results from the preferential formation of either triplet PG-3adLPG-PG, or paired PG-3adLPG complexes, dependent upon the mixing proportions of the two lipids. The relevance of these findings to the role native LPG in S. aureus, are discussed with respect to their influence on antibiotic resistance and lateral membrane organisation.


Asunto(s)
Liposomas/química , Lisina/química , Fosfatidilgliceroles/química , Staphylococcus aureus/metabolismo , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Microscopía por Crioelectrón , Liposomas/metabolismo , Lisina/metabolismo , Difracción de Neutrones , Fosfatidilgliceroles/metabolismo
4.
Molecules ; 25(24)2020 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-33371475

RESUMEN

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.


Asunto(s)
Aptámeros de Péptidos/metabolismo , Péptidos/metabolismo , Unión Proteica/fisiología , Aminoácidos/metabolismo , Anticuerpos/metabolismo , Sitios de Unión/fisiología , Comunicación Celular/fisiología , Hormonas/metabolismo , Ligandos , Conformación Molecular , Péptidos Cíclicos/metabolismo , Transducción de Señal/fisiología
5.
Commun Biol ; 3(1): 697, 2020 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-33247193

RESUMEN

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.


Asunto(s)
Antibacterianos/farmacología , Proteínas de Peces/farmacología , Enfermedades Pulmonares/tratamiento farmacológico , Proteínas Citotóxicas Formadoras de Poros/farmacología , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas de Peces/química , Proteínas de Peces/uso terapéutico , Células HEK293 , Células HeLa , Humanos , Enlace de Hidrógeno , Enfermedades Pulmonares/microbiología , Masculino , Membranas Artificiales , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Conformación Proteica
6.
ACS Pharmacol Transl Sci ; 3(3): 418-424, 2020 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-32566907

RESUMEN

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

7.
Anal Chem ; 91(23): 14865-14872, 2019 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-31660733

RESUMEN

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.


Asunto(s)
Calorimetría/métodos , Pruebas de Enzimas , Epóxido Hidrolasas/química , Compuestos Epoxi/química , Ácidos Grasos/química , Adamantano/análogos & derivados , Adamantano/química , Biocatálisis , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Ácidos Grasos/metabolismo , Análisis de Inyección de Flujo/métodos , Humanos , Hidrólisis , Cinética , Ácidos Láuricos/química , Metabolismo de los Lípidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluciones , Especificidad por Sustrato
8.
Sci Rep ; 9(1): 10934, 2019 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-31358802

RESUMEN

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.


Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Membrana Celular/efectos de los fármacos , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Transporte Iónico , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa , Liposomas Unilamelares/química
9.
Commun Biol ; 2: 188, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31123712

RESUMEN

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.


Asunto(s)
Epóxido Hidrolasas/antagonistas & inhibidores , Prostaglandina D2/análogos & derivados , Regulación Alostérica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico/genética , Cristalografía por Rayos X , Cisteína/química , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Prostaglandina D2/farmacología , Dominios Proteicos , Alineación de Secuencia , Solubilidad
10.
Nucleic Acids Res ; 47(8): 4272-4291, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-30820564

RESUMEN

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.


Asunto(s)
Autoantígenos/química , Poli A/química , Proteínas de Unión a Poli(A)/química , ARN Mensajero/química , Proteínas de Unión al ARN/química , Ribonucleoproteínas/química , Secuencias de Aminoácidos , Autoantígenos/genética , Autoantígenos/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Poli A/genética , Poli A/metabolismo , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Especificidad por Sustrato , Termodinámica , Antígeno SS-B
11.
Sci Rep ; 9(1): 1385, 2019 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-30718667

RESUMEN

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.


Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/metabolismo , Secuencia de Aminoácidos , Membrana Celular/efectos de los fármacos , Conductividad Eléctrica , Membrana Dobles de Lípidos/química , Micelas , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Conformación Proteica , Dodecil Sulfato de Sodio , Relación Estructura-Actividad
12.
Sci Rep ; 8(1): 17073, 2018 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-30451966

RESUMEN

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

13.
Sci Rep ; 6: 37639, 2016 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-27874065

RESUMEN

The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Bacterias Gramnegativas/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Ontología de Genes , Bacterias Gramnegativas/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/química , Simulación de Dinámica Molecular , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Transcriptoma/genética
14.
Sci Rep ; 6: 25182, 2016 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-27121989

RESUMEN

The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.


Asunto(s)
Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/patología , Mutación Puntual , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica
15.
Biochem J ; 473(1): 43-54, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26487699

RESUMEN

Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.


Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Glicoproteínas/metabolismo , Zinc/metabolismo , Adipoquinas , Sitios de Unión/fisiología , Proteínas Portadoras/química , Ácidos Grasos/química , Glicoproteínas/química , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Zinc/farmacología
16.
Nucleic Acids Res ; 43(1): 645-60, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25488812

RESUMEN

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3' UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5' UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.


Asunto(s)
Regiones no Traducidas 5' , Autoantígenos/química , Colágeno/genética , Ribonucleoproteínas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Autoantígenos/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Ribonucleoproteínas/genética , Alineación de Secuencia , Antígeno SS-B
17.
PLoS One ; 9(8): e105021, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25133504

RESUMEN

The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs) are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920) reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS), Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers) were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs).


Asunto(s)
Benzodiazepinas/química , ADN/química , G-Cuádruplex , Pirroles/química , Dicroismo Circular , Transferencia Resonante de Energía de Fluorescencia , Humanos , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray
18.
FEBS Lett ; 587(24): 3949-54, 2013 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-24188824

RESUMEN

Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the ß-adrenergic receptor.


Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Zinc/metabolismo , Adipoquinas , Sitios de Unión , Ácidos Grasos/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Estructura Secundaria de Proteína , Receptores Adrenérgicos beta/metabolismo , Especificidad por Sustrato , Zinc/química
19.
PLoS One ; 8(10): e76014, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24124530

RESUMEN

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.


Asunto(s)
Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteína p53 Supresora de Tumor/genética
20.
J Control Release ; 172(3): 929-38, 2013 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-24144917

RESUMEN

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.


Asunto(s)
Preparaciones de Acción Retardada/química , ARN Interferente Pequeño/administración & dosificación , beta-Alanina/análogos & derivados , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Endocitosis , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , beta-Alanina/química
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