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1.
Bioorg Chem ; 145: 107181, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38354503

RESUMEN

The human CC chemokine receptor 8 (CCR8) has been extensively pursued as target for the treatment of various inflammatory disorders. More recently, the importance of CCR8 in the tumor microenvironment has been demonstrated, spurring the interest in CCR8 antagonism as therapeutic strategy in immuno-oncology. On a previously described naphthalene sulfonamide with CCR8 antagonistic properties, the concept of isosterism was applied, leading to the discovery of novel CCR8 antagonists with IC50 values in the nM range in both the CCL1 competition binding and CCR8 calcium mobilization assay. The excellent CCR8 antagonistic activity of the most potent congeners was rationalized by homology molecular modeling.


Asunto(s)
Quimiocinas CC , Receptores de Quimiocina , Humanos , Quimiocinas CC/metabolismo , Quimiocina CCL1/metabolismo , Receptores de Quimiocina/química , Receptores de Quimiocina/metabolismo , Amidas , Receptores CCR8 , Sulfonamidas/farmacología , Naftalenos/farmacología
2.
Proteins ; 2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33713051

RESUMEN

Symmetric proteins are currently of interest as they allow creation of larger assemblies and facilitate the incorporation of metal ions in the larger complexes. Recently this was demonstrated by the biomineralization of the cadmium-chloride nanocrystal via the Pizza designer protein. However, the mechanism behind this formation remained unclear. Here, we set out to investigate the mechanism driving the formation of this nanocrystal via truncation, mutation, and circular permutations. In addition, the interaction of other biologically relevant metal ions with these symmetric proteins to form larger symmetric complexes was also studied. The formation of the initial nanocrystal is shown to originate from steric strain, where His 58 induces a different rotameric conformation on His 73, thereby distorting an otherwise perfect planar ring of alternating cadmium and chlorine ions, resulting in the smallest nanocrystal. Similar highly symmetric complexes were also observed for the other biological relevant metal ions. However, the flexibility of the coordinating histidine residues allows each metal ion to adopt its preferred geometry leading to either monomeric or dimeric ß-propeller units, where the metal ions are located at the interface between both propeller units. These results demonstrate that symmetric proteins are not only interesting to generate larger assemblies, but are also the perfect scaffold to create more complex metal based assemblies. Such metal protein assemblies may then find applications in bionanotechnology or biocatalysis.

3.
Bioorg Chem ; 107: 104560, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33383325

RESUMEN

The naphthalene sulfonamide scaffold is known to possess CCR8 antagonistic properties. In order to expand the structure-activity relationship study of this compound class, a variety of palladium-catalyzed cross-coupling reactions was performed on a bromo-naphthalene precursor yielding a diverse library. These compounds displayed CCR8 antagonistic properties in binding and calcium mobilization assays, with IC50 values in the 0.2 - 10 µM range. The decreased activity, when compared to the original lead compound, was rationalized by homology molecular modeling.


Asunto(s)
Bromo/química , Naftalenos/química , Paladio/química , Receptores CCR8/antagonistas & inhibidores , Sitios de Unión , Catálisis , Humanos , Simulación del Acoplamiento Molecular , Naftalenos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores CCR8/metabolismo , Relación Estructura-Actividad
4.
Proc Natl Acad Sci U S A ; 111(42): 15102-7, 2014 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-25288768

RESUMEN

The modular structure of many protein families, such as ß-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical ß-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.


Asunto(s)
Mycobacterium tuberculosis/enzimología , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Proteínas/química , Secuencia de Aminoácidos , Biofisica , Dicroismo Circular , Cristalografía por Rayos X , Luz , Modelos Moleculares , Modelos Teóricos , Datos de Secuencia Molecular , Nanotecnología , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray , Ultracentrifugación
5.
Angew Chem Int Ed Engl ; 54(34): 9857-60, 2015 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-26136355

RESUMEN

We have engineered a metal-binding site into the novel artificial ß-propeller protein Pizza. This new Pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. The designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. Two copies of the trimer associate in the presence of cadmium chloride in solution, and very high-resolution X-ray crystallographic analysis reveals a nanocrystal of cadmium chloride, sandwiched between two trimers of the protein. This nanocrystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. Our results indicate the feasibility of using rationally designed symmetrical proteins to biomineralize nanocrystals with useful properties.


Asunto(s)
Cloruro de Cadmio/química , Nanopartículas/química , Proteínas/química , Cristalografía por Rayos X , Modelos Moleculares , Ingeniería de Proteínas
6.
Anal Biochem ; 448: 92-4, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24333278

RESUMEN

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner.


Asunto(s)
Proteínas Portadoras/metabolismo , Luciferasas/metabolismo , Proteína SUMO-1/metabolismo , Espectrometría de Fluorescencia , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Genes Reporteros , Humanos , Luz , Luciferasas/química , Luciferasas/genética , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética
7.
J Comput Aided Mol Des ; 28(4): 363-73, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24446075

RESUMEN

The SAMPL challenges provide an ideal opportunity for unbiased evaluation and comparison of different approaches used in computational drug design. During the fourth round of this SAMPL challenge, we participated in the virtual screening and binding pose prediction on inhibitors targeting the HIV-1 integrase enzyme. For virtual screening, we used well known and widely used in silico methods combined with personal in cerebro insights and experience. Regular docking only performed slightly better than random selection, but the performance was significantly improved upon incorporation of additional filters based on pharmacophore queries and electrostatic similarities. The best performance was achieved when logical selection was added. For the pose prediction, we utilized a similar consensus approach that amalgamated the results of the Glide-XP docking with structural knowledge and rescoring. The pose prediction results revealed that docking displayed reasonable performance in predicting the binding poses. However, prediction performance can be improved utilizing scientific experience and rescoring approaches. In both the virtual screening and pose prediction challenges, the top performance was achieved by our approaches. Here we describe the methods and strategies used in our approaches and discuss the rationale of their performances.


Asunto(s)
Diseño Asistido por Computadora , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/enzimología , Simulación del Acoplamiento Molecular , Diseño de Fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Integrasa de VIH/química , Humanos , Unión Proteica , Programas Informáticos
8.
Sci Rep ; 11(1): 18867, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34552189

RESUMEN

[Formula: see text]-Propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats ('blades') each consisting of a 4-stranded anti-parallel [Formula: see text]-sheet. So far, 4- to 12-bladed [Formula: see text]-propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric [Formula: see text]-propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each [Formula: see text]-propeller sub-family.

9.
FEBS J ; 288(2): 530-545, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32343866

RESUMEN

ß-propeller proteins are common in nature, where they are observed to adopt 4- to 10-fold internal rotational pseudo-symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted ß-propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9-bladed ß-propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8-fold and 9-fold propellers which are highly stable. Cake variants with 2-10 identical copies of the repeat sequence were characterised by X-ray crystallography and in solution. They were found to be highly stable, and to self-assemble into 8- or 9-fold symmetrical propellers. These findings show that the ß-propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins. DATABASE: Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI.


Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Methanococcus/química , Ingeniería de Proteínas/métodos , Pseudomonas aeruginosa/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Methanococcus/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Termodinámica
10.
J Agric Food Chem ; 69(6): 1925-1935, 2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33533594

RESUMEN

Saccharomyces cerevisiae (S. cerevisiae)invertase is encoded by a family of closely related SUC genes. To identify and understand the molecular basis for differences in substrate specificity, we examined 29 SUC alleles from industrialS. cerevisiaestrains and cloned alleles with small sequence differences into an invertase-negative strain. Our study showed that an F102Y substitution in Suc-enzymes lowers yeast invertase activity toward fructo-oligosaccharides (FOS) by 36% and the specificity factor by 43%. By contrast, an A409P substitution in Suc-enzymes resulted in an increased capacity of the yeast to hydrolyze FOS and Fibruline by 17 and 41%, respectively, likely because of a change in the loop conformation resulting in a wider active site. Bread dough fermentation experiments revealed that sucrose and fructan hydrolysis during fermentation is influenced by this natural variation in SUC sequences. Our research thus opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that may ultimately allow controlling fructan hydrolysis.


Asunto(s)
Fructanos , Saccharomyces cerevisiae , Pan , Fermentación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo
11.
Comput Struct Biotechnol J ; 18: 3959-3968, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33335692

RESUMEN

Since the determination of the first molecular models of proteins there has been interest in creating proteins artificially, but such methods have only become widely successful in the last decade. Gradual improvements over a long period of time have now yielded numerous examples of non-natural proteins, many of which are built from repeated elements. In this review we discuss the design of such symmetrical proteins and their various applications in chemistry and medicine.

12.
Chem Commun (Camb) ; 56(78): 11601-11604, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32869783

RESUMEN

Novel bioinorganic hybrid materials based on proteins and inorganic clusters have enormous potential for the development of hybrid catalysts that synergistically combine properties of both materials. Here we report the creation of hybrid assemblies between a computationally designed symmetrical protein Pizza6-S and different polyoxometalates with matching symmetry: the tellurotungstic Anderson-Evans (Na6[TeW6O24]·22H2O) (TEW); Keggin (H4[SiW12O40]·6H2O) (STA); and 1 : 2 CeIII-substituted Keggin (K11[CeIII[PW11O39]2]·20H2O) (Ce-K). This results in the formation of complexes with clearly defined stoichiometries in solution. Crystal structures validate the complexes as building blocks for the formation of larger assemblies.


Asunto(s)
Proteínas/química , Compuestos de Tungsteno/química , Calorimetría , Cerio/química , Complejos de Coordinación/química , Cristalografía por Rayos X , Conformación Molecular , Unión Proteica , Ultracentrifugación
13.
J Struct Biol X ; 4: 100027, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32647829

RESUMEN

Recently an artificial protein named Pizza6 was reported, which possesses six identical tandem repeats and adopts a monomeric ß -propeller fold with sixfold structural symmetry. Pizza2, a truncated form that consists of a double tandem repeat, self-assembles into a trimer reconstructing the same propeller architecture as Pizza6. The ability of pizza proteins to self-assemble to form complete propellers makes them interesting building blocks to engineer larger symmetrical protein complexes such as symmetric nanoparticles. Here we have explored the self-assembly of Pizza2 fused to homo-oligomerizing peptides. In total, we engineered five different fusion proteins, of which three appeared to assemble successfully into larger complexes. Further characterization of these proteins showed one monodisperse designer protein with a structure close to the intended design. This protein was further fused to eGFP to investigate functionalization of the nanoparticle. The fusion protein was stable and could be expressed in high yield, showing that Pizza-based nanoparticles may be further decorated with functional domains.

14.
Protein Sci ; 29(12): 2375-2386, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33006397

RESUMEN

The ß-propeller fold is adopted by a sequentially diverse family of repeat proteins with apparent rotational symmetry. While the structure is mostly stabilized by hydrophobic interactions, an additional stabilization is provided by hydrogen bonds between the N-and C-termini, which are almost invariably part of the same ß-sheet. This feature is often referred to as the "Velcro" closure. The positioning of the termini within a blade is variable and depends on the protein family. In order to investigate the influence of this location on protein structure, folding and stability, we created different circular permutants, and a circularized version, of the designer propeller protein named Pizza. This protein is perfectly symmetrical, possessing six identical repeats. While all mutants adopt the same structure, the proteins lacking the "Velcro" closure were found to be significantly less resistant to thermal and chemical denaturation. This could explain why such proteins are rarely observed in nature. Interestingly the most common "Velcro" configuration for this protein family was not the most stable among the Pizza variants tested. The circularized version shows dramatically improved stability, which could have implications for future applications.


Asunto(s)
Modelos Moleculares , Pliegue de Proteína , Proteínas/química , Dicroismo Circular , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Proteínas/genética , Termodinámica
15.
Eur J Med Chem ; 205: 112638, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-32835918

RESUMEN

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.


Asunto(s)
Diseño de Fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/química , Pirimidinas/síntesis química , Pirimidinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Química Sintética , Humanos , Concentración 50 Inhibidora , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química
16.
J Enzyme Inhib Med Chem ; 24(3): 646-54, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18951281

RESUMEN

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40 degrees C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three beta-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no alpha-helix is present. The circular dichroism spectrum of TLXI confirms the absence of alpha-helices and the presence of antiparallel beta-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1-12 and for at least 2 hours at 100 degrees C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.


Asunto(s)
Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Triticum/química , Secuencia de Aminoácidos , Dicroismo Circular , Cisteína/química , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por Sustrato , Temperatura
17.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-31321399

RESUMEN

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Asunto(s)
Hidrolasas/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálisis , Cobre/química , Histidina/química , Histidina/genética , Hidrolasas/genética , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Nitrofenoles/química , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Treonina/química , Zinc/química
18.
IUCrJ ; 6(Pt 1): 46-55, 2019 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-30713702

RESUMEN

ß-Propeller proteins form one of the largest families of protein structures, with a pseudo-symmetrical fold made up of subdomains called blades. They are not only abundant but are also involved in a wide variety of cellular processes, often by acting as a platform for the assembly of protein complexes. WD40 proteins are a subfamily of propeller proteins with no intrinsic enzymatic activity, but their stable, modular architecture and versatile surface have allowed evolution to adapt them to many vital roles. By computationally reverse-engineering the duplication, fusion and diversification events in the evolutionary history of a WD40 protein, a perfectly symmetrical homologue called Tako8 was made. If two or four blades of Tako8 are expressed as single polypeptides, they do not self-assemble to complete the eight-bladed architecture, which may be owing to the closely spaced negative charges inside the ring. A different computational approach was employed to redesign Tako8 to create Ika8, a fourfold-symmetrical protein in which neighbouring blades carry compensating charges. Ika2 and Ika4, carrying two or four blades per subunit, respectively, were found to assemble spontaneously into a complete eight-bladed ring in solution. These artificial eight-bladed rings may find applications in bionanotechnology and as models to study the folding and evolution of WD40 proteins.

19.
Methods Mol Biol ; 1529: 309-322, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27914059

RESUMEN

Monomeric proteins with a number of identical repeats creating symmetrical structures are potentially very valuable building blocks with a variety of bionanotechnological applications. As such proteins do not occur naturally, the emerging field of computational protein design serves as an excellent tool to create them from nonsymmetrical templates. Existing pseudo-symmetrical proteins are believed to have evolved from oligomeric precursors by duplication and fusion of identical repeats. Here we describe a computational workflow to reverse-engineer this evolutionary process in order to create stable proteins consisting of identical sequence repeats.


Asunto(s)
Biología Computacional/métodos , Evolución Molecular , Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/genética , Secuencia de Aminoácidos , Secuencia Conservada , Bases de Datos Genéticas , Programas Informáticos
20.
Sci Rep ; 7(1): 5943, 2017 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-28724971

RESUMEN

Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial ß-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galß(1,4)Glc) on the surface, but the cytotoxicity is abolished.


Asunto(s)
Lectinas/química , Neoplasias/metabolismo , Neoplasias/patología , Factores Trefoil/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Muerte Celular , Línea Celular Tumoral , Biología Computacional , Cristalografía por Rayos X , Hemaglutinación , Humanos , Lectinas/metabolismo , Peso Molecular , Dominios Proteicos , Multimerización de Proteína , Conejos , Azúcares/metabolismo
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