RESUMEN
Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing Cre discrimination between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the plasticity of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations were used to investigate the possible mechanisms behind the specificity switch. Our findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.
Asunto(s)
Integrasas , Recombinación Genética , Integrasas/genética , Integrasas/metabolismo , Integrasas/química , Especificidad por Sustrato , Mutación , ADN/química , ADN/genética , ADN Intergénico/genética , ADN Intergénico/química , Evolución Molecular Dirigida/métodosRESUMEN
Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Edición Génica , Ingeniería Genética/métodos , Recombinación Genética , Células HEK293 , HumanosRESUMEN
Angiogenesis is an important physiological process playing a crucial role in wound healing and cancer progression. Vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are key players in angiogenesis. Based on previous findings regarding the modulation of VEGF activity by glycosaminoglycans (GAG), here we explore the interaction of hyaluronan (HA)-based GAG with PDGF and its receptor PDGFR-ß by applying molecular modeling and dynamics simulations in combination with surface plasmon resonance (SPR). Computational analysis on the interaction of oligo-hyaluronan derivatives with different sulfation pattern and functionalization shows that these GAG interact with PDGF in relevant regions for receptor recognition, and that high sulfation as well as modification with the TAMRA group convey stronger binding. On the other hand, the studied oligo-hyaluronan derivatives are predicted to scarcely recognize PDGFR-ß. SPR results are in line with the computational predictions regarding the binding pattern of HA tetrasaccharide (HA4) derivatives to PDGF and PDGFR-ß. Furthermore, our experimental results also show that the complexation of PDGF to PDGFR-ß can be modulated by HA4 derivatives. The results found open the path for considering HA4 derivatives as potential candidates to be exploited for modulation of the PDGF/PDGFR-ß signaling system in angiogenesis and related disease conditions.
Asunto(s)
Ácido Hialurónico/química , Factor de Crecimiento Derivado de Plaquetas/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Conformación de Carbohidratos , Humanos , Modelos Moleculares , Proteínas Recombinantes/química , Resonancia por Plasmón de SuperficieRESUMEN
The chemokine interleukin-8 (IL-8, CXCL8) plays an important role in inflammatory processes and consecutive wound healing. It recruits primarily neutrophils to infection sites and stimulates their degranulation and phagocytosis in effector cells. IL-8 binds glycosaminoglycans (GAGs), a class of complex linear anionic polysaccharides often organized into diversely sulfated micro-domains, that enriches the protein concentration locally and so facilitate the formation of stable concentration gradients. In this study, we applied experimental and computational techniques to investigate the binding of wild type and truncated IL-8 variants to natural and chemically modified GAGs to gain further insight into the IL-8/GAG interaction. Circular dichroism spectroscopy of IL-8 variants did not reveal major structural changes upon GAG binding. Heparin affinity chromatography clearly demonstrates that gradual truncation of the C-terminal helix leads to decreasing affinities. Similarly, surface plasmon resonance indicates participation of both IL-8 termini in GAG binding, which strength is dependent on GAG sulfation degree. Molecular modeling suggests that C-terminal truncation of IL-8 weakens the interaction with GAGs by an alteration of IL-8 GAG binding site. Our study provides more detailed understanding of the IL-8/GAG interaction and contributes to the data of potential use for the development of biomedical implications in tissue regeneration.
Asunto(s)
Glicosaminoglicanos/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mutación/genética , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Heparina/química , Interleucina-8/química , Unión Proteica , Receptores de Interleucina-8A , Regeneración , TermodinámicaRESUMEN
Tyrosine-type site-specific recombinases (T-SSRs) have opened new avenues for the predictable modification of genomes as they enable precise genome editing in heterologous hosts. These enzymes are ubiquitous in eubacteria, prevalent in archaea and temperate phages, present in certain yeast strains, but barely found in higher eukaryotes. As tools they find increasing use for the generation and systematic modification of genomes in a plethora of organisms. If applied in host organisms, they enable precise DNA cleavage and ligation without the gain or loss of nucleotides. Criteria directing the choice of the most appropriate T-SSR system for genetic engineering include that, whenever possible, the recombinase should act independent of cofactors and that the target sequences should be long enough to be unique in a given genome. This review is focused on recent advancements in our mechanistic understanding of simple T-SSRs and their application in developmental and synthetic biology, as well as in biomedical research.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Integrasas/metabolismo , Tirosina/metabolismo , ADN/metabolismo , ADN Nucleotidiltransferasas/química , Integrasas/química , Conformación ProteicaRESUMEN
It is well recognized that high molecular weight hyaluronan (H-HA) exerts potent anti-inflammatory effects while its fragmentation into low molecular weight HA (L-HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti-inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL-HA). However, the molecular mechanisms by which sL-HA produces its anti-inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL-HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL-HA did not affect cell viability but resulted in a reduced pro-inflammatory cytokine response of iMФ after activation indicating a profound counter-regulation of their initial inflammatory phenotype. Rapid internalization of sL-HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL-HA after activation whereas anti-inflammatory proteins were induced. This study proves anti-inflammatory properties of sL-HA and provides information on its regulatory mode of action on iMФ.
RESUMEN
Glycosaminoglycans (GAGs) exhibit a key role in cellular communication processes through interactions with target proteins of the extracellular matrix (ECM). The sandwich-like interaction established between Fibroblast growth factor (FGF) and heparin (HE) represents quite a peculiar protein-GAG-protein system, which has been both structurally and functionally intensively studied. The molecular recognition characteristics of this system have been exploited in various computational studies in order to deepen understanding of GAG-protein interactions. Here, we drill down on the interactions established in this peculiar macromolecular complex by analyzing the applicability of docking techniques and molecular dynamics (MD)-based approaches, and we dissect the molecular recognition properties exhibited by FGF towards a series of HE derivatives. We examine the sensitivity of MM-GBSA free energy calculations in terms of receptor conformational space sampling and changes in the ligand structures. Furthermore, we investigate its predictive power in combination with other computational methods, namely the well-established Autodock3 (AD3) and dynamic molecular docking (DMD), a targeted MD-based docking method specifically developed to account for flexibility and solvent in computer simulations of protein-GAG systems. Our results show that a site-mapping approach can be effectively combined with AD3 and DMD calculations to accurately reproduce available experimental data and, furthermore, to determine specific GAG recognition patterns. This study deepens our understanding of the applicability of available theoretical approaches to the investigation of molecular recognition in protein-GAG systems.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/química , Heparina/química , Simulación del Acoplamiento Molecular , Sulfatos/química , Secuencias de Aminoácidos , Sitios de Unión , Secuencia de Carbohidratos , Cristalografía por Rayos X , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , TermodinámicaRESUMEN
Glycosaminoglycans represent a class of linear anionic periodic polysaccharides, which play a key role in a variety of biological processes in the extracellular matrix via interactions with their protein targets. Computationally, glycosaminoglycans are very challenging due to their high flexibility, periodicity and electrostatics-driven nature of the interactions with their protein counterparts. In this work, we carry out a detailed computational characterization of the interactions in protein-glycosaminoglycan complexes from the Protein Data Bank (PDB), which are split into two subsets accounting for their intrinsic nature: non-enzymatic-protein-glycosaminoglycan and enzyme-glycosaminoglycan complexes. We apply molecular dynamics to analyze the differences in these two subsets in terms of flexibility, retainment of the native interactions in the simulations, free energy components of binding and contributions of protein residue types to glycosaminoglycan binding. Furthermore, we systematically demonstrate that protein electrostatic potential calculations, previously found to be successful for glycosaminoglycan binding sites prediction for individual systems, are in general very useful for proposing protein surface regions as putative glycosaminoglycan binding sites, which can be further used for local docking calculations with these particular polysaccharides. Finally, the performance of six different docking programs (Autodock 3, Autodock Vina, MOE, eHiTS, FlexX and Glide), some of which proved to perform well for particular protein-glycosaminoglycan complexes in previous work, is evaluated on the complete protein-glycosaminoglycan data set from the PDB. This work contributes to widen our knowledge of protein-glycosaminoglycan molecular recognition and could be useful to steer a choice of the strategies to be applied in theoretical studies of these systems.
Asunto(s)
Biología Computacional/métodos , Glicosaminoglicanos/química , Simulación del Acoplamiento Molecular , Proteínas/química , Sitios de Unión , Bases de Datos de Proteínas , Glicosaminoglicanos/metabolismo , Cinética , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/metabolismo , Electricidad Estática , TermodinámicaRESUMEN
The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.
Asunto(s)
Quimiocina CXCL12/química , Glicosaminoglicanos/química , Sitios de Unión , Conformación de Carbohidratos , Humanos , Modelos MolecularesRESUMEN
Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.
Asunto(s)
Glicosaminoglicanos/administración & dosificación , Ácido Hialurónico/administración & dosificación , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Inhibidor Tisular de Metaloproteinasa-3/biosíntesis , Sulfatos de Condroitina/administración & dosificación , Sulfatos de Condroitina/química , Endocitosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/química , Humanos , Ácido Hialurónico/química , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Células Madre Mesenquimatosas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Resonancia por Plasmón de Superficie , Inhibidor Tisular de Metaloproteinasa-3/química , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The recruitment of different chemokines and growth factors by glycosaminoglycans (GAGs) such as chondroitin sulfate or hyaluronan plays a critical role in wound healing processes. Thus, there is a special interest in the design of artificial extracellular matrices with improved properties concerning GAG interaction with common regulating proteins. In this study, amide hydrogen/deuterium (H/D) exchange mass spectrometry (HDX MS) combined with molecular modeling and docking experiments was used to obtain structural models of proinflammatory chemokine interleukin-8 (IL-8) in complex with hexameric chondroitin sulfate. Experiments on the intact protein showed a difference in deuterium labeling of IL-8 due to chondroitin sulfate binding. The extent of deuteration was reduced from 24% to 13% after 2 min exchange time, which corresponds to a reduced exchange of approximately 10 backbone amides. By local HDX MS experiments, H/D exchange information on the complete sequence of IL-8 could be obtained. A significantly reduced H/D exchange, especially of the C-terminal α-helical region comprising amino acids 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and chondroitin sulfate determined this way correlated excellently with the corresponding NMR based atomistic model previously published. Our results demonstrate that HDX-MS in combination with molecular modeling is a valuable approach for the analysis of protein/GAG complexes at physiological pH, temperature, and salt concentration. The fact that HDX-MS requires only micrograms of protein and GAGs makes it a very promising technique to address protein-GAG interactions.
Asunto(s)
Amidas/química , Medición de Intercambio de Deuterio/métodos , Glicosaminoglicanos/análisis , Hidrógeno/química , Interleucina-8/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Catepsina K/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Líquido del Lavado Bronquioalveolar , Dicroismo Circular , Inhibidores de Cisteína Proteinasa/farmacología , Fibrosis Quística/microbiología , Humanos , Macrófagos Alveolares/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Especificidad por Sustrato , CatelicidinasRESUMEN
The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a ßI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a ß-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An N(ε)-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by (1)H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired ßI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.
Asunto(s)
Colágeno/química , Péptidos/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Cromatografía en Capa Delgada , Dicroismo Circular , Modelos Moleculares , Conformación MolecularRESUMEN
Glycosaminoglycans (GAGs) represent a class of anionic periodic linear polysaccharides, which mediate cell communication processes by interactions with their protein targets in the extracellular matrix. Due to their high flexibility, charged nature, periodicity, and polymeric nature, GAGs are challenging systems for computational approaches. To deal with the length challenge, coarse-grained (CG) modeling could be a promising approach. In this work, we develop AMBER-compatible CG parameters for GAGs using all-atomic (AA) molecular dynamics (MD) simulations in explicit solvent and the Boltzmann conversion approach. We compare both global and local properties of GAGs obtained in the simulations with AA and CG approaches, and we conclude that our CG model is appropriate for the MD approach of long GAG molecules at long time scales.
Asunto(s)
Glicosaminoglicanos/química , Simulación de Dinámica Molecular , Biología Computacional/métodos , Glicosaminoglicanos/metabolismo , Heparina/química , Enlace de Hidrógeno , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , SolventesRESUMEN
The Cre/loxP system is widely used as a genetic tool to manipulate DNA. Cre recombinase catalyzes site-specific recombination between 34 bp loxP sites. Each loxP site is recognized by two Cre molecules assuming a cleaving (CreC) and non-cleaving (CreNC) activity. Despite the symmetry in the sequences of the arms of loxP, available biochemical data show strong evidence that the recombination reaction is asymmetric with a preferred strand exchange order. The asymmetry comes from the spacer separating the two sets of palindromic arms of the loxP sequence. However, it remains to be understood how this preferential order is established. We apply computational structure-based methods and perform a thorough detailed analysis of available structural and biochemical information on the Cre/loxP system in order to investigate such asymmetry in the recombination, and we propose a rationale to explain the determinants favoring the strand exchange order. We show that the structural properties of the DNA flanking sequence of the spacer guide the recombination, and we establish the role of residues R118, R121 and K122 from CreC, which contact the spacer region and by clamping the DNA inhibit the cleavage on the second arm of loxP. Our studies give an atomistic insight on the synapsis state of the recombination process in the Cre/loxP system and highlight the importance of the flexibility and other intrinsic properties of the flanking regions of the DNA spacer to establish a preferential strand exchange order.
Asunto(s)
ADN Intergénico , Integrasas/química , Simulación de Dinámica Molecular , Secuencias de Aminoácidos , ADN Intergénico/química , ADN Intergénico/metabolismo , Integrasas/metabolismo , Modelos Moleculares , Conformación Proteica , Recombinación GenéticaRESUMEN
Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.
Asunto(s)
Evolución Molecular Dirigida/métodos , Recombinasas/química , Sitios de Ligazón Microbiológica , ADN/química , ADN/metabolismo , Modelos Moleculares , Unión Proteica , Recombinasas/genética , Recombinasas/metabolismoRESUMEN
Numerous biological processes (tissue formation, remodelling and healing) are strongly influenced by the cellular microenvironment. Glycosaminoglycans (GAGs) are important components of the native extracellular matrix (ECM) able to interact with biological mediator proteins. They can be chemically functionalized and thereby modified in their interaction profiles. Thus, they are promising candidates for functional biomaterials to control healing processes in particular in health-compromised patients. Biophysical studies show that the interaction profiles between mediator proteins and GAGs are strongly influenced by (i) sulphation degree, (ii) sulphation pattern, and (iii) composition and structure of the carbohydrate backbone. Hyaluronan derivatives demonstrate a higher binding strength in their interaction with biological mediators than chondroitin sulphate for a comparable sulphation degree. Furthermore sulphated GAG derivatives alter the interaction profile of mediator proteins with their cell receptors or solute native interaction partners. These results are in line with biological effects on cells relevant for wound healing processes. This is valid for solute GAGs as well as those incorporated in collagen-based artificial ECM (aECMs). Prominent effects are (i) anti-inflammatory, immunomodulatory properties towards macrophages/dendritic cells, (ii) enhanced osteogenic differentiation of human mesenchymal stromal cells, (iii) altered differentiation of fibroblasts to myofibroblasts, (iv) reduced osteoclast activity and (v) improved osseointegration of dental implants in minipigs. The findings of our consortium Transregio 67 contribute to an improved understanding of structure-function relationships of GAG derivatives in their interaction with mediator proteins and cells. This will enable the design of bioinspired, functional biomaterials to selectively control and promote bone and skin regeneration.
Asunto(s)
Materiales Biocompatibles , Glicosaminoglicanos/química , Animales , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Modelos Animales , Resonancia por Plasmón de SuperficieRESUMEN
The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.
Asunto(s)
Glicosaminoglicanos/metabolismo , Interleucina-10/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Animales , Ratones , Simulación de Dinámica MolecularRESUMEN
Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.
Asunto(s)
Proteína Morfogenética Ósea 2/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Glicosaminoglicanos/química , Materiales Biocompatibles/química , Técnicas Biosensibles , Sulfatos de Condroitina/química , Biología Computacional , Matriz Extracelular/química , Humanos , Ácido Hialurónico/química , Conformación Proteica , Termodinámica , Ingeniería de TejidosRESUMEN
We present Dynamic Molecular Docking (DMD), a novel targeted molecular dynamics-based protocol developed to address ligand and receptor flexibility as well as the inclusion of explicit solvent in local molecular docking. A class of ligands for which docking performance especially benefits from overcoming these challenges is the glycosaminoglycans (GAGs). GAGs are periodic, highly flexible, and negatively charged polysaccharides playing an important role in the extracellular matrix via interaction with proteins such as growth factors and chemokines. The goal of our work has been to develop a proof of concept for an MD-based docking approach and to analyze its applicability for protein-GAG systems. DMD exploits the electrostatics-driven attraction of a ligand to its receptor, treats both as entirely flexible, and considers solvent explicitly. We show that DMD has high predictive significance for systems dominated by electrostatic attraction and demonstrate its capability to reliably identify the receptor residues contributing most to binding.