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1.
Plant Cell ; 34(9): 3400-3424, 2022 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-35640532

RESUMEN

For most Gram-negative bacteria, pathogenicity largely depends on the type-III secretion system that delivers virulence effectors into eukaryotic host cells. The subcellular targets for the majority of these effectors remain unknown. Xanthomonas campestris, the causal agent of black rot disease of crucifers such as Brassica spp., radish, and turnip, delivers XopP, a highly conserved core-effector protein produced by X. campestris, which is essential for virulence. Here, we show that XopP inhibits the function of the host-plant exocyst complex by direct targeting of Exo70B, a subunit of the exocyst complex, which plays a significant role in plant immunity. XopP interferes with exocyst-dependent exocytosis and can do this without activating a plant NOD-like receptor that guards Exo70B in Arabidopsis. In this way, Xanthomonas efficiently inhibits the host's pathogen-associated molecular pattern (PAMP)-triggered immunity by blocking exocytosis of pathogenesis-related protein-1A, callose deposition, and localization of the FLAGELLIN SENSITIVE2 (FLS2) immune receptor to the plasma membrane, thus promoting successful infection. Inhibition of exocyst function without activating the related defenses represents an effective virulence strategy, indicating the ability of pathogens to adapt to host defenses by avoiding host immunity responses.


Asunto(s)
Arabidopsis , Xanthomonas campestris , Proteínas Bacterianas , Enfermedades de las Plantas , Inmunidad de la Planta , Virulencia
2.
Plant J ; 116(1): 100-111, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37344990

RESUMEN

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium-dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Xanthomonas campestris , Xanthomonas campestris/metabolismo , Arabidopsis/metabolismo , Fosforilación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
3.
Nature ; 521(7553): 541-544, 2015 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-25799992

RESUMEN

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.


Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas Mad2/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Recombinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Humanos , Cambio de Clase de Inmunoglobulina/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mad2/deficiencia , Proteínas Mad2/genética , Ratones , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/metabolismo
4.
Bioorg Med Chem Lett ; 29(18): 2626-2631, 2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31362921

RESUMEN

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Pargilina/farmacología , Pirrolina Carboxilato Reductasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Pargilina/síntesis química , Pargilina/química , Pirrolina Carboxilato Reductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , delta-1-Pirrolina-5-Carboxilato Reductasa
5.
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
6.
Proc Natl Acad Sci U S A ; 112(5): 1505-10, 2015 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-25605945

RESUMEN

MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.


Asunto(s)
Antígenos de Histocompatibilidad Clase I/química , Péptidos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Entropía , Cinética , Péptidos/inmunología
7.
J Immunol ; 193(10): 4803-13, 2014 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-25311806

RESUMEN

Virus or tumor Ag-derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.


Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Antígeno HLA-A2/inmunología , Neoplasias/prevención & control , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfocitos B , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/química , Cristalografía por Rayos X , Epítopos , Expresión Génica , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Humanos , Inmunización , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/inmunología , Péptidos/administración & dosificación , Péptidos/química , Péptidos/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo
8.
J Struct Biol ; 179(1): 46-55, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22580066

RESUMEN

Baculovirus infected insect cells are widely used for heterologous protein expression. Despite the power of this system, the use of baculovirus techniques for protein expression screening is hampered by the time and resources needed to generate each recombinant baculovirus. Here, we show that a transfection/infection based expression system is suitable for screening of expression constructs in insect cells and represents a valid alternative to other traditional screening methodologies using recombinant baculovirus. The described method is based on gene delivery by transfection coupled to the induction of protein expression by non-recombinant baculovirus infection. Vectors that control expression by a combination of the baculovirus promoters ie1 and p10 and the enhancer element hr5 are among the ones suitable for this method. Infection with non-recombinant baculovirus drastically increases the basal activity of these elements, leading to protein over-expression. Multiple vectors can be simultaneously co-transfected/infected, making transfection/infection amenable for screening of multiple co-expressed proteins and protein complexes. Taken together, our results prove that the transfection/infection protocol is a valid and innovative approach for increasing speed and reducing costs of protein expression screening for structural and functional studies.


Asunto(s)
Baculoviridae/genética , Vectores Genéticos/genética , Proteínas Recombinantes/biosíntesis , Spodoptera/virología , Transfección/métodos , Animales , Baculoviridae/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Spodoptera/citología , Spodoptera/metabolismo
9.
J Struct Biol ; 175(2): 113-9, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21453775

RESUMEN

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.


Asunto(s)
Clonación Molecular/métodos , Endopeptidasas/genética , Vectores Genéticos , Proteínas Recombinantes/genética , Animales , Automatización de Laboratorios , Baculoviridae , Secuencia de Bases , Línea Celular , Endopeptidasas/metabolismo , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Proteasas Ubiquitina-Específicas
10.
J Struct Biol ; 175(2): 159-70, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21382497

RESUMEN

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.


Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Vectores Genéticos/normas , Complejos Multiproteicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Academias e Institutos , Factor de Unión a CCAAT/biosíntesis , Factor de Unión a CCAAT/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Europa (Continente) , Geminina , Cooperación Internacional , Israel , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción TFII/biosíntesis , Factores de Transcripción TFII/genética
11.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 12): 1545-7, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22139162

RESUMEN

Crinumin, a novel glycosylated serine protease with chymotrypsin-like catalytic specificity, was purified from the medicinally important plant Crinum asiaticum. Crinumin is a 67.7 kDa protease with an extraordinary stability and activity over a wide range of pH and temperature and is functional in aqueous, organic and chaotropic solutions. The purified protease has thrombolytic and antiplatelet activity. The use of C. asiaticum extracts has also been reported for the treatment of a variety of disorders such as injury, joint inflammation and arthritis. In order to understand its structure-function relationship, the enzyme was purified from the plant latex and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from a single crystal and processed to 2.8 Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 121.61, b = 95.00, c = 72.10 Å, α = γ = 90, ß = 114.19°. The Matthews coefficient was 2.81 Å(3) Da(-1), corresponding to a solvent content of 56%, assuming one molecule in the asymmetric unit. Structure determination of the enzyme is in progress.


Asunto(s)
Anticoagulantes/química , Crinum/enzimología , Fibrinolíticos/química , Proteínas de Plantas/química , Serina Proteasas/química , Anticoagulantes/metabolismo , Cristalización , Cristalografía por Rayos X , Fibrinolíticos/metabolismo , Glicosilación , Proteínas de Plantas/metabolismo , Serina Proteasas/metabolismo
12.
Nat Struct Mol Biol ; 12(7): 582-8, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15951818

RESUMEN

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.


Asunto(s)
Proteínas Portadoras/química , Conotoxinas/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Caracoles/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Conotoxinas/genética , Conotoxinas/farmacología , Cristalografía , Electrofisiología , Humanos , Datos de Secuencia Molecular , Mutación/genética , Neuronas/metabolismo , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Oocitos/metabolismo , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Xenopus
13.
J Am Chem Soc ; 131(34): 12298-304, 2009 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-19655750

RESUMEN

High-throughput structure determination of protein-ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, "conditional", peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This "in crystallo" exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC beta2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families.


Asunto(s)
Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Rayos Ultravioleta , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica/efectos de la radiación , Conformación Proteica/efectos de la radiación , Selenio/química
14.
J Am Chem Soc ; 131(34): 12305-13, 2009 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-19655751

RESUMEN

Class I major histocompatibility complexes (MHCs) present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. The unstable nature of unliganded MHC necessitates the production of recombinant class I complexes through in vitro refolding reactions in the presence of an added excess of peptides. This strategy is not amenable to high-throughput production of vast collections of class I complexes. To address this issue, we recently designed photocaged MHC ligands that can be cleaved by a UV light trigger in the MHC bound state under conditions that do not affect the integrity of the MHC structure. The results obtained with photocaged MHC ligands demonstrate that conditional MHC ligands can form a generally applicable concept for the creation of defined peptide-MHCs. However, the use of UV exposure to mediate ligand exchange is unsuited for a number of applications, due to the lack of UV penetration through cell culture systems and due to the transfer of heat upon UV irradiation, which can induce evaporation. To overcome these limitations, here, we provide proof-of-concept for the generation of defined peptide-MHCs by chemical trigger-induced ligand exchange. The crystal structure of the MHC with the novel chemosensitive ligand showcases that the ligand occupies the expected binding site, in a conformation where the hydroxyl groups should be reactive to periodate. We proceed to validate this technology by producing peptide-MHCs that can be used for T cell detection. The methodology that we describe here should allow loading of MHCs with defined peptides in cell culture devices, thereby permitting antigen-specific T cell expansion and purification for cell therapy. In addition, this technology will be useful to develop miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands.


Asunto(s)
Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Ácido Peryódico/química , Ácido Peryódico/farmacología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Epítopos/inmunología , Humanos , Cinética , Ligandos , Modelos Moleculares , Péptidos/metabolismo , Procesos Fotoquímicos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Linfocitos T/inmunología , Linfocitos T/metabolismo , Técnicas de Cultivo de Tejidos , Rayos Ultravioleta
15.
Nat Struct Mol Biol ; 26(7): 567-570, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31270470

RESUMEN

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Humanos , Simulación del Acoplamiento Molecular , Conformación Proteica , Dominios Proteicos , Tubulina (Proteína)/metabolismo
16.
Neuron ; 41(6): 907-14, 2004 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-15046723

RESUMEN

Nicotinic acetylcholine receptors are prototypes for the pharmaceutically important family of pentameric ligand-gated ion channels. Here we present atomic resolution structures of nicotine and carbamylcholine binding to AChBP, a water-soluble homolog of the ligand binding domain of nicotinic receptors and their family members, GABAA, GABAC, 5HT3 serotonin, and glycine receptors. Ligand binding is driven by enthalpy and is accompanied by conformational changes in the ligand binding site. Residues in the binding site contract around the ligand, with the largest movement in the C loop. As expected, the binding is characterized by substantial aromatic and hydrophobic contributions, but additionally there are close contacts between protein oxygens and positively charged groups in the ligands. The higher affinity of nicotine is due to a main chain hydrogen bond with the B loop and a closer packing of the aromatic groups. These structures will be useful tools for the development of new drugs involving nicotinic acetylcholine receptor-associated diseases.


Asunto(s)
Acetilcolina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Activación del Canal Iónico/fisiología , Agonistas Nicotínicos/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Carbacol/metabolismo , Proteínas Portadoras/efectos de los fármacos , Cristalografía por Rayos X , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Lymnaea , Modelos Moleculares , Conformación Molecular , Sistema Nervioso/metabolismo , Nicotina/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología
17.
J Mol Neurosci ; 30(1-2): 9-10, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17192605

RESUMEN

Acetylcholine-binding protein (AChBP) is a water-soluble protein released from molluscan glial cells and modulates ACh-mediated synaptic transmission. Acetylcholine-binding protein (AChBP) is a water-soluble homolog of the ligand-binding domain of nicotinic receptors and other members of the pharmaceutically important family of pentameric ligand-gated ion channels (LGICs), GABAA, GABAC, 5-HT3 serotonin, and glycine receptors. The crystal structure of AChBP from Lymnaea stagnalis has become an established model for the extracellular domain of the pentameric LGICs, and homology models have been generated to analyze receptor-ligand interactions. AChBP has pharmacological properties similar to the homomeric alpha7 subtype of nicotinic ACh receptors (nAChRs), with relatively weak affinity for ACh and a 10-fold higher affinity for nicotine.


Asunto(s)
Proteínas Portadoras/fisiología , Lymnaea/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Conotoxinas/química , Venenos de Moluscos/química , Conformación Proteica , Caracoles
18.
FEBS Lett ; 585(22): 3593-9, 2011 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-22036717

RESUMEN

The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors.


Asunto(s)
Antineoplásicos/química , Pirimidinas/química , Receptor EphA4/química , Tiazoles/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dasatinib , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Receptor EphA4/antagonistas & inhibidores
19.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 10): 1232-42, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17001100

RESUMEN

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.


Asunto(s)
Células Eucariotas/metabolismo , Células Procariotas/metabolismo , Proteínas Recombinantes/biosíntesis , Algoritmos , Animales , Seguridad Computacional , Simulación por Computador , Quinasas Ciclina-Dependientes/metabolismo , Reparación del ADN , Bases de Datos Genéticas , Escherichia coli/metabolismo , Vectores Genéticos , Gestión de la Información , Insectos/metabolismo , ARN/biosíntesis , ARN/genética , Receptores Citoplasmáticos y Nucleares/genética , Factor de Transcripción TFIID/genética , Ubiquitina-Proteína Ligasas/genética , Quinasa Activadora de Quinasas Ciclina-Dependientes
20.
J Biol Chem ; 280(28): 26457-66, 2005 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-15899893

RESUMEN

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-HT3), gamma-aminobutyric acid (GABA), types A and C, and glycine receptors. Here we present the crystal structure of a remote homolog, AChBP from Bulinus truncatus, which reveals both the conserved structural scaffold and the sites of variation in this receptor family. These include rigid body movements of loops that are close to the transmembrane interface in the receptors and changes in the intermonomer contacts, which alter the pentamer stability drastically. Structural, pharmacological and mutational analysis of both AChBPs shows how 3 amino acid changes in the binding site contribute to a 5-10-fold difference in affinity for nicotinic ligands. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling, and drug design.


Asunto(s)
Proteínas Portadoras/química , Receptores Colinérgicos/química , Receptores Nicotínicos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bungarotoxinas/química , Calorimetría , Dicroismo Circular , Clonación Molecular , Cristalografía por Rayos X , Diseño de Fármacos , Hibridación in Situ , Iones , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Moluscos , Filogenia , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Temperatura
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