The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.
- MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- molekulární struktura MeSH
- responzivní elementy MeSH
- transkripční faktory chemie metabolismus MeSH
- vodík-deuteriová výměna MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.
- MeSH
- bezlepková dieta * MeSH
- celiakie enzymologie imunologie terapie MeSH
- Drosophila metabolismus MeSH
- enzymoterapie * MeSH
- gliadin metabolismus MeSH
- gluteny metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- myši inbrední NOD MeSH
- myši MeSH
- proteiny vázající GTP metabolismus MeSH
- proteolýza MeSH
- transglutaminasy metabolismus MeSH
- zánět imunologie metabolismus prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.
As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.
- MeSH
- buněčná inkluze MeSH
- disulfidy chemie MeSH
- Escherichia coli metabolismus MeSH
- hmotnostní spektrometrie MeSH
- lektinové receptory NK-buněk - podrodina B biosyntéza chemie genetika MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- refolding proteinů MeSH
- rekombinantní proteiny biosyntéza MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein.
- MeSH
- aspartátové proteasy chemie genetika metabolismus MeSH
- disulfidy MeSH
- koncentrace vodíkových iontů MeSH
- Magnoliopsida enzymologie genetika MeSH
- masožravci MeSH
- redukční činidla MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rychlý rozvoj badatelského výzkumu v moderní glykobiochemii a glykobiologii vedl k širokému zpřístupnění nových experimentálních postupů přejímaných vzápětí biotechnologickým průmyslem pro produkci glykoproteinových terapeutik a diagnostik. Pokrok posledního období je založen na důkladných a systematických znalostech detailů enzymové mašinérie vedoucí ke specifické glykosylaci proteinů, která zůstává jednou z nejčastějších a nejdramatičtějších ko- a posttranslačních modifikací proteinů. Klonování genů kódujících jednotlivé glykosylační enzymy umožnilo jejich použití v buněčných technologiích a buněčném inženýrství vedoucím k široké dostupnosti linií produkujících spolehlivě humánní typ glykosylace. Pokrok v těchto technikách probíhal paralelně s rozvojem metod umožňujících provést komplexní analýzu proteinové glykosylace, a byl nyní rozsáhle využit biotechnologickými firmami k produkci více než dvou stovek schválených terapeutických proteinů, z nichž má více než polovina charakter glykoproteinů.
Rapid progress in contemporary glycobiology led to widespread availability of new technologies employed by biotechnology industry for production of glycoprotein therapeutics and diagnostics. This progress has been based on our detailed knowledge of enzyme machinery enabling protein glycosylation, one of the most often and most extensive posttranslational protein modification. Cloning of individual genes coding for glycosylation enzymes enabled their use in cellular technologies and cellular engineering resulting in wide availability of cell lines able to produce human glycosylation efficiently and precisely. Progress in this direction went in parallel with that in the techniques suitable for analytical evaluation of glycosylation, and has been now widely adopted at biotechnological companies producing more then two hundred therapeutical proteins, more then half of them being glycosylated.
The glycan code of glycoproteins can be conceptually defined at molecular level by the sequence of well characterized glycans attached to evolutionarily predetermined amino acids along the polypeptide chain. Functional consequences of protein glycosylation are numerous, and include a hierarchy of properties from general physicochemical characteristics such as solubility, stability and protection of the polypeptide from the environment up to specific glycan interactions. Definition of the glycan code for glycoproteins has been so far hampered by the lack of chemically defined glycoprotein glycoforms that proved to be extremely difficult to purify from natural sources, and the total chemical synthesis of which has been hitherto possible only for very small molecular species. This review summarizes the recent progress in chemical and chemoenzymatic synthesis of complex glycans and their protein conjugates. Progress in our understanding of the ways in which a particular glycoprotein glycoform gives rise to a unique set of functional properties is now having far reaching implications for the biotechnology of important glycodrugs such as therapeutical monoclonal antibodies, glycoprotein hormones, carbohydrate conjugates used for vaccination and other practically important protein-carbohydrate conjugates.
- MeSH
- biotechnologie metody MeSH
- glykoproteiny biosyntéza chemie metabolismus MeSH
- glykosylace MeSH
- konformace sacharidů MeSH
- molekulární sekvence - údaje MeSH
- oligosacharidy chemie metabolismus MeSH
- pojmy organické chemie MeSH
- polysacharidy chemická syntéza chemie metabolismus MeSH
- sacharidové sekvence MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.
- MeSH
- alfa-N-acetylgalaktosaminidasa biosyntéza chemie genetika MeSH
- Aspergillus niger enzymologie genetika MeSH
- buněčné kultury MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- elektronová mikroskopie MeSH
- gelová chromatografie MeSH
- glykosylace MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- rekombinantní proteiny biosyntéza chemie genetika MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glucose allowing to produce homogenous soluble monomeric NKp30 in several formats needed for advanced NMR studies. Our optimized protocol now allows to produce routinely 10mg batches of these NKp30ex proteins per 1L of M9 production medium in four working days. The purity and identity of the produced proteins were checked by SDS-PAGE, MALDI MS peptide mapping, and high resolution ion cyclotron resonance MS. Analytical ultracentrifugation confirmed the monomeric status of the produced proteins. Long-term stability of the produced protein proved to be very good allowing its use for NMR studies using elevated temperatures. These studies should reveal further details of the interaction of NKp30 with several of its ligands including target cell surface proteins and heparin-derived oligosaccharides.
- MeSH
- bioreaktory MeSH
- chlorid amonný chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- Escherichia coli chemie genetika metabolismus MeSH
- kodon MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- receptor 3 spouštějící přirozenou cytotoxicitu biosyntéza chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- ultracentrifugace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
