RESUMO
The diterpene cafestol represents the most potent cholesterol-elevating compound known in the human diet, being responsible for more than 80% of the effect of coffee on serum lipids, with a mechanism still not fully clarified. In the present study, the interaction of cafestol and 16-O-methylcafestol with the stabilized ligand-binding domain (LBD) of the Farnesoid X Receptor was evaluated by fluorescence and circular dichroism. Fluorescence quenching was observed with both cafestol and 16-O-methylcafestol due to an interaction occurring in the close environment of the tryptophan W454 residue of the protein, as confirmed by docking and molecular dynamics. A conformational change of the protein was also observed by circular dichroism, particularly for cafestol. These results provide evidence at the molecular level of the interactions of FXR with the coffee diterpenes, confirming that cafestol can act as an agonist of FXR, causing an enhancement of the cholesterol level in blood serum.
Assuntos
Colesterol , Café , Diterpenos , Receptores Citoplasmáticos e Nucleares , Diterpenos/farmacologia , Diterpenos/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Colesterol/metabolismo , Humanos , Café/química , Simulação de Acoplamento Molecular , Ligação Proteica , Simulação de Dinâmica Molecular , Dicroísmo CircularRESUMO
Inclusion of polymethine cyanine dyes in the cavity of macrocyclic receptors is an effective strategy to alter their absorption and emission behavior in aqueous solution. In this paper, the effect of the host-guest interaction between cucurbit[8]uril (CB[8]) and a model trimethine indocyanine (Cy3) on dye spectral properties and aggregation in water is investigated. Solution studies, performed by a combination of spectroscopic and calorimetric techniques, indicate that the addition of CB[8] disrupts Cy3 aggregates, leading to the formation of a 1 : 1 host-guest complex with an association constant of 1.5×106 â M-1 . At concentrations suitable for NMR experiments, the slow formation of a supramolecular polymer was observed, followed by precipitation. Single crystals X-ray structure elucidation confirmed the formation of a polymer with 1 : 1 stoichiometry in the solid state.
Assuntos
Hidrocarbonetos Aromáticos com Pontes , Quinolinas , Hidrocarbonetos Aromáticos com Pontes/química , Corantes , Compostos Heterocíclicos com 2 Anéis , Imidazolidinas , Compostos Macrocíclicos , Espectroscopia de Ressonância Magnética , Polímeros , Água/químicaRESUMO
Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient-derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases.
Assuntos
DNA Helicases/genética , Anemia de Fanconi/genética , Instabilidade Genômica/genética , Síndrome de Kearns-Sayre/metabolismo , Miopatias Mitocondriais/metabolismo , Anormalidades Múltiplas/genética , RNA Helicases DEAD-box/genética , DNA Helicases/metabolismo , Anemia de Fanconi/metabolismo , Genômica , Humanos , Síndrome de Kearns-Sayre/genética , Miopatias Mitocondriais/genética , Mutação/genéticaRESUMO
Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.
Assuntos
Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Ligação Competitiva , Cisteína/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Oxirredutases/química , Oxirredutases/genética , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Estabilidade Proteica , Receptor ErbB-2/química , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/química , Anticorpos de Domínio Único/químicaRESUMO
Bone defects are a global health concern; bone tissue engineering (BTE) is the most promising alternative to reduce patient morbidity and overcome the inherent drawbacks of autograft and allograft bone. Three-dimensional scaffolds are pivotal in this field due to their potential to provide structural support and mimic the natural bone microenvironment. Following an already published protocol, a 3D porous structure consisting of alginate and hydroxyapatite was prepared after a gelation step and a freezing-drying step. Despite the frequent use of alginate in tissue regeneration, the biological inertness of this polysaccharide hampers proper cell colonization and proliferation. Therefore, the purpose of this work was to enhance the biological properties by promoting the interaction and adhesion between cells and biomaterial with the use of Fibronectin. This extracellular matrix protein was physically adsorbed on the scaffold, and its presence was evaluated with environmental scanning electron microscopy (eSEM) and the Micro-Bicinchoninic Acid (µBCA) protein assay. The MG-63 cell line was used for both static and dynamic (i.e., in bioreactor) 3D cell culturing on the scaffolds. The use of the bioreactor allowed for a better exchange of nutrients and oxygen and a better removal of cell catabolites from the inner portion of the construct, mimicking the physiological environment. The functionalized scaffolds showed an improvement in cell proliferation and colonization compared to non-functionalized ones; the effect of the addition of Fibronectin was more evident in the dynamic culturing conditions, where the cells clearly adhered on the surface of functionalized scaffolds.
RESUMO
In bone regeneration, combining natural polymer-based scaffolds with Bioactive Glasses (BGs) is an attractive strategy to improve the mechanical properties of the structure, as well as its bioactivity and regenerative potential. Methods: For this purpose, a well-studied alginate/hydroxyapatite (Alg/HAp) porous scaffold was enhanced with an experimental bioglass (BGMS10), characterized by a high crystallization temperature and containing therapeutic ions such as strontium and magnesium. This resulted in an improved biological response compared to 45S5 Bioglass®, the "gold" standard among BGs. Porous composite scaffolds were fabricated by freeze-drying technique and characterized by scanning electron microscopy and microanalysis, infrared spectroscopy, and microcomputed tomography. The mechanical properties and cytocompatibility of the new scaffold composition were also evaluated. The addition of bioglass to the Alg/HAp network resulted in a slightly lower porosity. However, despite the change in pore size, the MG-63 cells were able to better adhere and proliferate when cultured for one week on a BG scaffold compared to the control Alg/HAp scaffolds. Thus, our findings indicate that the combination of bioactive glass BGMS10 does not affect the structural and physicochemical properties of the Alg/HAp scaffold and confers bioactive properties to the structures, making the Alg/HAp-BGMS10 scaffold a promising candidate for future application in bone tissue regeneration.
RESUMO
Hexameric helicases are responsible for many biological processes, ranging from DNA replication in various life domains to DNA repair, transcriptional regulation and RNA metabolism, and encompass superfamilies 3-6 (SF3-6).To harness the chemical energy from ATP hydrolysis for mechanical work, hexameric helicases have a conserved core engine, called ASCE, that belongs to a subdivision of the P-loop NTPases. Some of the ring helicases (SF4 and SF5) use a variant of ASCE known as RecA-like, while some (SF3 and SF6) use another variant known as AAA+ fold. The NTP-binding sites are located at the interface between monomers and include amino-acid residues coming from neighbouring subunits, providing a mean for small structural changes within the ATP-binding site to be amplified into large inter-subunit movement.The ring structure has a central channel which encircles the nucleic acid. The topological link between the protein and the nucleic acid substrate increases the stability and processivity of the enzyme. This is probably the reason why within cellular systems the critical step of unwinding dsDNA ahead of the replication fork seems to be almost invariably carried out by a toroidal helicase, whether in bacteria, archaea or eukaryotes, as well as in some viruses.Over the last few years, a large number of biochemical, biophysical and structural data have thrown new light onto the architecture and function of these remarkable machines. Although the evidence is still limited to a couple of systems, biochemical and structural results suggest that motors based on RecA and AAA+ folds have converged on similar mechanisms to couple ATP-driven conformational changes to movement along nucleic acids.
Assuntos
DNA Helicases , Replicação do DNA , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , DNA/química , DNA Helicases/química , Nucleosídeo-Trifosfatase , Estrutura Terciária de ProteínaRESUMO
Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes.
Assuntos
Anticorpos de Domínio Único , Afinidade de Anticorpos , Anticorpos de Domínio Único/química , Epitopos , Proteínas Recombinantes/genéticaRESUMO
The eukaryotic MCM2-7 complex is recruited at origins of replication during the G1 phase and acts as the main helicase at the replication fork during the S phase of the cell cycle. To characterize the interplay between the MCM helicase and DNA prior to the melting of the double helix, we determined the structure of an archaeal MCM orthologue bound to a 5.6-kb double-stranded DNA segment, using cryo-electron microscopy. DNA wraps around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. Our findings provide novel insights into the role of the MCM complex during the initiation step of DNA replication.
Assuntos
Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/ultraestrutura , DNA/ultraestrutura , Methanobacteriaceae/enzimologia , Sítios de Ligação , Modelos Moleculares , Proteínas Mutantes/ultraestrutura , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid. At variance with conventional surface plasmon resonance detection, MGEC-TLS characterization confirmed the capability of the assay to titrate the HER2-ECD antigen down to concentrations of 440 ng/mL. The high versatility of the directed protein-DNA conjugates immobilization through DNA hybridization on plasmonic scaffolds and coupled with the high sensitivity of the MGEC-TLS detection qualifies the proposed assay as a potential, easily operated biosensing strategy for the fast and label-free detection of disease-relevant antigens.
RESUMO
The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal ß-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.
RESUMO
In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains.
Assuntos
Camelídeos Americanos/genética , Mutação , Anticorpos de Domínio Único/química , Animais , Camelídeos Americanos/imunologia , Clonagem Molecular , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. In parallel, the complex was cross-linked, digested and the resulting peptides were characterized by mass-spectrometry to define maximal distance restraints between HER2 and A10 amino acids in their complex. These independent datasets guided a docking process, refined by molecular dynamics simulations, to develop a model of the complex and estimate per-residue free-energy contributions. Such a model explains the experimental data and identifies a second, non-canonical paratope, located in the region opposite to the conventional nanobody paratope, formed by the hypervariable loop regions LH1 and LH3. Both paratopes contributed substantially to the overall affinity by binding to independent HER2 epitopes. Nanobody mutants with substitution of key interaction residues, as indicated by the model, possess significantly lower affinity for HER2. This is the first described case of a "natural" biparatopic nanobody, directly selected by in-vitro panning.
Assuntos
Sítios de Ligação de Anticorpos , Receptor ErbB-2/química , Anticorpos de Cadeia Única/química , Humanos , Simulação de Acoplamento Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
We present an in silico mutagenetic protocol for improving the binding affinity of single domain antibodies (or nanobodies, VHHs). The method iteratively attempts random mutations in the interacting region of the protein and evaluates the resulting binding affinity towards the target by scoring, with a collection of scoring functions, short explicit solvent molecular dynamics trajectories of the binder-target complexes. The acceptance/rejection of each attempted mutation is carried out by a consensus decision-making algorithm, which considers all individual assessments derived from each scoring function. The method was benchmarked by evolving a single complementary determining region (CDR) of an anti-HER2 VHH hit obtained by direct panning of a phage display library. The optimised VHH mutant showed significantly enhanced experimental affinity with respect to the original VHH it matured from. The protocol can be employed as it is for the optimization of peptides, antibody fragments, and (given enough computational power) larger antibodies.
Assuntos
Reações Antígeno-Anticorpo , Simulação por Computador , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Algoritmos , Humanos , Simulação de Dinâmica Molecular , Mutação , Receptor ErbB-2/imunologia , Anticorpos de Domínio Único/genéticaRESUMO
GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia Eletrônica , Modelos Moleculares , Multimerização Proteica , Espalhamento a Baixo ÂnguloRESUMO
The tumor suppressor inhibitor of growth 4 (ING4) regulates chromatin structure by recruiting the histone acetyl transferase complex HBO1 to sites with histone H3 trimethylated at K4. ING4 dimerizes through its N-terminal domain and recognizes H3K4me3 by the C-terminal plant homeodomain (PHD). The central region of ING4 is disordered and contains the nuclear localization signal. Here, utilizing electrophoresis and nuclear magnetic resonance, we show that ING4 binds double-stranded DNA through its central region with micromolar affinity. Our findings suggest that the cooperativity arising from the presence of two DNA-binding regions in the ING4 dimer, as well as two H3K4me3-binding PHD fingers, may strengthen nucleosome binding and HBO1 complex recruitment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Fenômenos Biofísicos , Proteínas de Ciclo Celular/química , Ensaio de Desvio de Mobilidade Eletroforética , Fluorescência , Proteínas de Homeodomínio/química , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Titulometria , Proteínas Supressoras de Tumor/químicaRESUMO
High-fidelity replication guarantees the stable inheritance of genetic information stored in the DNA of living organisms. The minichromosome maintenance (MCM) complex functions as replicative DNA-unwinding helicase and has been identified as one key player in the replication process of archea and eukarya. Despite the availability of considerable structural information on archeal MCMs, such information was missing for their C-terminal domain. In order to obtain more detailed structural information, we assigned the NMR chemical shifts for backbone and side chain nuclei for the MCM C-terminal winged helix domains of the archeal species Methanothermobacter thermautrophicus and Sulfolobus solfataricus.
Assuntos
Methanobacteriaceae/enzimologia , Proteínas de Manutenção de Minicromossomo/química , Ressonância Magnética Nuclear Biomolecular , Sulfolobus solfataricus/enzimologia , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.