RESUMO
Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.
Assuntos
Surfactantes Pulmonares , Síndrome do Desconforto Respiratório do Recém-Nascido , Síndrome do Desconforto Respiratório , Recém-Nascido , Animais , Feminino , Coelhos , Adulto , Humanos , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Surfactantes Pulmonares/farmacologia , Surfactantes Pulmonares/uso terapêutico , Surfactantes Pulmonares/química , Tensoativos/uso terapêutico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Peptídeos/farmacologia , Peptídeos/químicaRESUMO
Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived proteins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naïve affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recognition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide technology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.
Assuntos
Biblioteca de Peptídeos , Engenharia de Proteínas , Citometria de Fluxo/métodos , Engenharia de Proteínas/métodos , Ligação ProteicaRESUMO
The exploration of therapies combining antimicrobial lung proteins and conventional antibiotics is important due to the growing problem of multidrug-resistant bacteria. The aim of this study was to investigate whether human SP-A and a recombinant trimeric fragment (rfhSP-A) have cooperative antimicrobial activity with antibiotics against pathogenic Gram-negative bacteria. We found that SP-A bound the cationic peptide polymyxin B (PMB) with an apparent dissociation constant (K D) of 0.32 ± 0.04 µM. SP-A showed synergistic microbicidal activity with polymyxin B and E, but not with other antibiotics, against three SP-A-resistant pathogenic bacteria: Klebsiella pneumoniae, non-typable Haemophilus influenzae (NTHi), and Pseudomonas aeruginosa. SP-A was not able to bind to K. pneumoniae, NTHi, or to mutant strains thereof expressing long-chain lipopolysaccharides (or lipooligosaccharides) and/or polysaccharide capsules. In the presence of PMB, SP-A induced the formation of SP-A/PMB aggregates that enhance PMB-induced bacterial membrane permeabilization. Furthermore, SP-A bound to a molecular derivative of PMB lacking the acyl chain (PMBN) with a K D of 0.26 ± 0.02 µM, forming SP-A/PMBN aggregates. PMBN has no bactericidal activity but can bind to the outer membrane of Gram-negative bacteria. Surprisingly, SP-A and PMBN showed synergistic bactericidal activity against Gram-negative bacteria. Unlike native supratrimeric SP-A, the trimeric rfhSP-A fragment had small but significant direct bactericidal activity against K. pneumoniae, NTHi, and P. aeruginosa. rfhSP-A did not bind to PMB under physiological conditions but acted additively with PMB and other antibiotics against these pathogenic bacteria. In summary, our results significantly improve our understanding of the antimicrobial actions of SP-A and its synergistic action with PMB. A peptide based on SP-A may aid the therapeutic use of PMB, a relatively cytotoxic antibiotic that is currently being reintroduced into clinics due to the global problem of antibiotic resistance.
Assuntos
Polimixina B , Polimixinas , Antibacterianos/química , Antibacterianos/farmacologia , Antibióticos Antineoplásicos , Bactérias , Bactérias Gram-Negativas/metabolismo , Humanos , Klebsiella pneumoniae , Polimixina B/metabolismo , Polimixina B/farmacologia , Polimixinas/química , Polimixinas/metabolismo , Polimixinas/farmacologia , Pseudomonas aeruginosa , Proteína A Associada a Surfactante PulmonarRESUMO
Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Ligação Proteica , Domínios Proteicos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aß42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit essentially identical ability to inhibit Aß42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells. We identified 70 proteins that had more significant interactions with rh Bri2 BRICHOS relative to the corresponding control particles. Three previously identified Bri2 BRICHOS interacting proteins were also identified in our 'fishing' experiments. The binding affinity of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the top 'hit', was calculated and was identified as a strong interacting partner. Enrichment analysis of the retained proteins identified three biological pathways: Rho GTPase, heat stress response and pyruvate, cysteine and methionine metabolism.
Assuntos
Peptídeos beta-Amiloides , Proteínas de Transporte , Proteínas Adaptadoras de Transdução de Sinal , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Humanos , Fenômenos Magnéticos , Ligação Proteica , ProteômicaRESUMO
Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer's disease-related amyloid-ß peptide (Aß42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aß42 but has a high affinity for Aß42 fibrils, using surface plasmon resonance. Kinetic analysis of Aß42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aß42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aß42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aß42 aggregates that are comprised of eight or fewer Aß42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.
Assuntos
Trifosfato de Adenosina/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Chaperonas Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas , Multimerização Proteica , Proteína C Associada a Surfactante Pulmonar/metabolismo , Humanos , Domínios Proteicos , Proteína C Associada a Surfactante Pulmonar/genéticaRESUMO
Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation. á .
Assuntos
Metilaminas/química , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Gases/química , Humanos , Íons/química , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de ProteínaRESUMO
Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide ß17. The fusion protein NT*-ß17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that ß17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, ß17 adopts a ß-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Fibroínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/química , Cálcio/metabolismo , Escherichia coli/genética , Fibroínas/química , Fibroínas/genética , Concentração de Íons de Hidrogênio , Ligação Proteica , Conformação Proteica , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sais/química , SolubilidadeRESUMO
Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
Assuntos
Proteínas Recombinantes/biossíntese , Seda/biossíntese , Tensoativos/química , Animais , Colecistocinina/química , Cromatografia , Dicroísmo Circular , Dimerização , Modelos Animais de Doenças , Escherichia coli/metabolismo , Feminino , Fibroínas/biossíntese , Concentração de Íons de Hidrogênio , Pulmão/patologia , Espectroscopia de Ressonância Magnética , Micelas , Microscopia Eletrônica de Transmissão , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Domínios Proteicos , Coelhos , Transtornos Respiratórios/tratamento farmacológico , AranhasRESUMO
The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.
Assuntos
Neoplasias/metabolismo , Receptor ErbB-3/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Alanina , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Dicroísmo Circular , Feminino , Humanos , Concentração Inibidora 50 , Ligantes , Camundongos , Mutação , Biblioteca de Peptídeos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Receptor ErbB-3/química , Receptor ErbB-3/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
Assuntos
Afinidade de Anticorpos/imunologia , Biblioteca de Peptídeos , Receptor ErbB-3/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Afinidade de Anticorpos/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , StaphylococcusRESUMO
Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host ( approximately 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
Assuntos
Citometria de Fluxo/métodos , Biblioteca de Peptídeos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus/genética , Staphylococcus/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Técnicas Biossensoriais , Dicroísmo Circular , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fator de Necrose Tumoral alfa/químicaRESUMO
The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.