RESUMO
Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.
Assuntos
Microscopia Crioeletrônica , Tireoglobulina/química , Tireoglobulina/ultraestrutura , Proteínas de Bactérias/química , Células HEK293 , Humanos , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Mutação , Reprodutibilidade dos Testes , Solventes/química , Tireoglobulina/genética , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMO
Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.
Assuntos
Proteínas Desgrenhadas/química , Receptores Frizzled/química , Proteínas Wnt/química , beta Catenina/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Clonagem Molecular , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The Chip/LIM-domain binding protein (LDB)-single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/metabolismo , Complexos Multiproteicos/química , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Wnt/genéticaRESUMO
We introduce a new family of fungal protease inhibitors with ß-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal ß-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the ß2-ß3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with ß-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with ß-trefoil fold.
Assuntos
Agaricales , Ácido Aspártico Proteases , Cisteína Proteases , Lotus , Agaricales/química , Ácido Aspártico Endopeptidases , Ácido Aspártico Proteases/genética , Cisteína , Cisteína Proteases/genética , Lotus/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/químicaRESUMO
The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both ß-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the ß2-ß3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the ß11-ß12 loop. Cnispin is a substrate-like inhibitor and the ß11-ß12 loop is yet another ß-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the ß2-ß3 and ß11-ß12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the ß2-ß3 loop in cnispin and into the ß11-ß12 loop in cospin. These results show that ß-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.
RESUMO
At present, the determination of crystal structures from data that have been acquired from twinned crystals is routine; however, with the increasing number of crystal structures additional crystal lattice disorders are being discovered. Here, a previously undescribed partial rotational order-disorder that has been observed in crystals of stefin B is described. The diffraction images revealed normal diffraction patterns that result from a regular crystal lattice. The data could be processed in space groups I4 and I422, yet one crystal exhibited a notable rejection rate in the higher symmetry space group. An explanation for this behaviour was found once the crystal structures had been solved and refined and the electron-density maps had been inspected. The lattice of stefin B crystals is composed of five tetramer layers: four well ordered layers which are followed by an additional layer of alternatively placed tetramers. The presence of alternative positions was revealed by the inspection of electron-density score maps. The well ordered layers correspond to the crystal symmetry of space group I422. In addition, the positions of the molecules in the additional layer are related by twofold rotational axes which correspond to space group I422; however, these molecules lie on the twofold axis and can only be related in a statistical manner. When the occupancies of alternate positions and overlapping are equal, the crystal lattice indeed fulfills the criteria of space group I422; when these occupancies are not equal, the lattice only fulfills the criteria of space group I4.
Assuntos
Cistatina B/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcß1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its ß-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.
Assuntos
Lactose/análogos & derivados , Ricina/química , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/genética , Sistema ABO de Grupos Sanguíneos/metabolismo , Agaricales , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Eritrócitos/química , Eritrócitos/metabolismo , Escherichia coli/genética , Humanos , Células Jurkat , Lactose/química , Lactose/genética , Lactose/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Ricina/genética , Ricina/metabolismo , Ricina/toxicidadeRESUMO
Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a ß-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the ß2 and ß3 strands, distinguishing cospin from other ß-trefoil-fold serine protease inhibitors in which ß4-ß5 or ß5-ß6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.
Assuntos
Agaricales/química , Carpóforos/química , Proteínas Fúngicas/química , Inibidores da Tripsina/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Assuntos
Catepsinas/química , Catepsinas/fisiologia , Sequência de Aminoácidos , Animais , Catepsinas/genética , Catepsinas/metabolismo , Biologia Celular/tendências , Cisteína Proteases/química , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Cisteína Proteases/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The ß-trefoil protease inhibitors that come from plants, first discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable ß-trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the ß-trefoil proteins a promising starting point for the generation of strong, specific, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modified properties for applications in many fields of medicine, biotechnology, and agriculture.
Assuntos
Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Inibidores de Proteases/metabolismo , Estrutura Secundária de ProteínaRESUMO
Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes ß-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.
Assuntos
Catepsina E/química , Sequência de Aminoácidos , Catepsina E/genética , Catepsina E/metabolismo , Escherichia coli/genética , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Microscopia de Força Atômica , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).
Assuntos
Antibacterianos/farmacologia , Compostos de Benzilideno/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Rodanina/análogos & derivados , Inibidores da Topoisomerase II , Antibacterianos/síntese química , Antibacterianos/química , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/química , DNA Girase/metabolismo , Relação Dose-Resposta a Droga , Enterococcus faecalis/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Haemophilus influenzae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Rodanina/síntese química , Rodanina/química , Rodanina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a beta-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded beta-barrel, and two layers of loops (6 + 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.
Assuntos
Inibidores de Cisteína Proteinase/química , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Catepsinas/química , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Proteínas Fúngicas/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Estrutura Secundária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Tripsina/metabolismoRESUMO
Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). We have identified an interaction between stefin B and nucleosomes, specifically with histones H2A.Z, H2B, and H3. In synchronized T98G cells, stefin B co-immunoprecipitated with histone H3, predominantly in the G(1) phase of the cell cycle. Stefin B-deficient mouse embryonic fibroblasts entered S phase earlier than wild type mouse embryonic fibroblasts. In contrast, increased expression of stefin B in the nucleus delayed cell cycle progression in T98G cells. The delay in cell cycle progression was associated with the inhibition of cathepsin L in the nucleus, as judged from the decreased cleavage of the CUX1 transcription factor. In vitro, inhibition of cathepsin L by stefin B was potentiated in the presence of histones, whereas histones alone did not affect the cathepsin L activity. Interaction of stefin B with the Met-75 truncated form of cathepsin L in the nucleus was confirmed by fluorescence resonance energy transfer experiments in the living cells. Stefin B could thus play an important role in regulating the proteolytic activity of cathepsin L in the nucleus, protecting substrates such as transcription factors from its proteolytic processing.
Assuntos
Catepsina L/metabolismo , Núcleo Celular/metabolismo , Cistatina B/metabolismo , Regulação da Expressão Gênica , Histonas/química , Animais , Ciclo Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Histonas/metabolismo , Humanos , Camundongos , Modelos BiológicosRESUMO
Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the ß-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses.
Assuntos
Proteína Axina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Histidina/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Proteína Axina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Retroalimentação , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologiaRESUMO
The cysteine protease inhibitors, clitocypin and macrocypins, from higher fungi (mycocypins), together with the serine protease inhibitors highly specific for trypsin cospin and cnispin from higher fungi (mycospins), display several characteristics that distinguish them from protease inhibitors from other sources. Their high genetic heterogeneity affects their functionality and/or stability and results in numerous protein variants with slightly different inhibitory profiles that influence the type of protease inhibited and/or the strength of inhibition. They possess the µ-trefoil fold that shows high plasticity in their utilization of the 11 diverse loops for the inhibition of various families of proteases through different mechanisms of inhibition. Their high versatility is also seen in their regulatory and defence functions and in their potential applications in biotechnology, crop protection and medicine.
RESUMO
Peptidoglycan is a giant molecule that forms the cell wall that surrounds bacterial cells. It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues connected by ß-(1,4)-glycosidic bonds and cross-linked with short polypeptide chains. Owing to the increasing antibiotic resistance against drugs targeting peptidoglycan synthesis, studies of enzymes involved in the degradation of peptidoglycan, such as N-acetylglucos-aminidases, may expose new, valuable drug targets. The scientific challenge addressed here is how lysozymes, muramidases which are likely to be the most studied enzymes ever, and bacterial N-acetylglucosaminidases discriminate between two glycosidic bonds that are different in sequence yet chemically equivalent in the same NAG-NAM polymers. In spite of more than fifty years of structural studies of lysozyme, it is still not known how the enzyme selects the bond to be cleaved. Using macromolecular crystallography, chemical synthesis and molecular modelling, this study explains how these two groups of enzymes based on an equivalent structural core exhibit a difference in selectivity. The crystal structures of Staphylococcus aureusN-acetylglucosaminidase autolysin E (AtlE) alone and in complex with fragments of peptidoglycan revealed that N-acetylglucosaminidases and muramidases approach the substrate at alternate glycosidic bond positions from opposite sides. The recognition pocket for NAM residues in the active site of N-acetylglucosaminidases may make them a suitable drug target.
RESUMO
Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins.
Assuntos
Proteínas de Bactérias/química , Parede Celular/metabolismo , Clostridioides difficile/metabolismo , Sítios de Ligação , Clostridioides difficile/química , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Cysteine cathepsins often contribute to cancer progression due to their overexpression in the tumour microenvironment and therefore present attractive targets for non-invasive diagnostic imaging. However, the development of highly selective and versatile small molecule probes for cathepsins has been challenging. Here, we targeted tumour-associated cathepsin B using designed ankyrin repeat proteins (DARPins). The selective DARPin 8h6 inhibited cathepsin B with picomolar affinity (Ki = 35 pM) by binding to a site with low structural conservation in cathepsins, as revealed by the X-ray structure of the complex. DARPin 8h6 blocked cathepsin B activity in tumours ex vivo and was successfully applied in in vivo optical imaging in two mouse breast cancer models, in which cathepsin B was bound to the cell membrane or secreted to the extracellular milieu by tumour and stromal cells. Our approach validates cathepsin B as a promising diagnostic and theranostic target in cancer and other inflammation-associated diseases.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Catepsina B/análise , Microscopia Intravital/métodos , Técnicas de Sonda Molecular , Animais , Catepsina B/química , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Camundongos , Ligação Proteica , Conformação ProteicaRESUMO
UNLABELLED: Lectins are carbohydrate-binding proteins present in all organisms. Some cytoplasmic lectins from fruiting bodies of dikaryotic fungi are toxic against a variety of parasites and predators. We have isolated, cloned and expressed a novel, single domain lectin from Macrolepiota procera, designated MpL. Determination of the crystal structure revealed that MpL is a ricin B-like lectin with a ß-trefoil fold. Biochemical characterization, site-directed mutagenesis, co-crystallization with carbohydrates, isothermal titration calorimetry and glycan microarray analyses show that MpL forms dimers with the carbohydrate-binding site at the α-repeat, with the highest specificity for terminal N-acetyllactosamine and other ß-galactosides. A second putative carbohydrate-binding site with a low affinity for galactose is present at the γ-repeat. In addition, a novel hydrophobic binding site was detected in MpL with specificity for molecules other than carbohydrates. The tissue specific distribution of MpL in the stipe and cap tissue of fruiting bodies and its toxicity towards the nematode Caenorhabditis elegans indicate a function of MpL in protecting fruiting bodies against predators and parasites. DATABASE: Nucleotide sequence data have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers HQ449738 and HQ449739. Structural data have been deposited in the Protein Data Bank under accession codes 4ION, 4IYB, 4IZX and 4J2S.