Polyglutamylation is a reversible posttranslational modification that is catalyzed by enzymes of the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation that is initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Dishevelled 3 (DVL3), thereby changing the interactome of DVL3. Polyglutamylation increases the capacity of DVL3 to get phosphorylated, to undergo phase separation, and to act in the noncanonical Wnt pathway. Both carboxy-terminal polyglutamylation and the resulting reduction in phase separation capacity of DVL3 can be reverted by the deglutamylating enzyme CCP6, demonstrating a causal relationship between TTLL11-mediated polyglutamylation and phase separation. Thus, C-terminal polyglutamylation represents a new type of posttranslational modification, broadening the range of proteins that can be modified by polyglutamylation and providing the first evidence that polyglutamylation can modulate protein phase separation.
- Klíčová slova
- Dishevelled 3, Noncanonical Wnt Signaling, Polyglutamylation, Protein Condensates, TTLL11,
- MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- kyselina polyglutamová metabolismus analogy a deriváty MeSH
- lidé MeSH
- peptidsynthasy * metabolismus genetika MeSH
- posttranslační úpravy proteinů * MeSH
- protein dishevelled * metabolismus genetika MeSH
- separace fází MeSH
- signální dráha Wnt MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DVL3 protein, human MeSH Prohlížeč
- kyselina polyglutamová MeSH
- peptidsynthasy * MeSH
- protein dishevelled * MeSH
- tubulin polyglutamylase MeSH Prohlížeč
Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.
- Klíčová slova
- Bacillaceae, Mucilage protein, Peribacillus simplex, Polyglutamic acid, Soil bacteria,
- MeSH
- Bacillus subtilis metabolismus MeSH
- Bacillus * metabolismus MeSH
- fylogeneze MeSH
- kyselina polyglutamová * analýza metabolismus MeSH
- polysacharidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kyselina polyglutamová * MeSH
- polysacharidy MeSH
In protein-based formulations, conformational distortions and attractive interactions may cause insoluble and undesired aggregates. In the case of ionic peptides, including cationic or anionic, commonly electrostatic interactions are the main factors that control structure assembling. In this study, it was proposed that grafting of chitosan (CS) to γ-polyglutamic acid (γ-PGA) might exhibit much strong inhibiting effect on the formation of protein aggregates due to multiple amino groups and hydrophilic properties. To guarantee stable and safe biopharmaceutical formulation, the potency of a variety of stabilizers including sugars (glucose, sucrose), polyols (sorbitol, glycerol), surfactant (Tween 20), salting-out salt (PBS), and also different pH values have been evaluated on stabilizing or destabilizing the native state of CS-g-PGA copolymer using FTIR, CD, DLS, and SDS-PAGE. The comparable analysis revealed that the stability of CS-g-PGA was strongly dependent on pH owing to the polyelectrolyte characteristics of the polymers. Altogether these results implied that CS at optimized conditions might be an important precursor for the pharmaceutical industry and function as a new polymer for aggregation suppression and protein stabilization.
- Klíčová slova
- Chemical stabilization, Chitosan, Protein folding, pH depending structure, γ-polyglutamic acid,
- MeSH
- chitosan * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina polyglutamová * chemie analogy a deriváty MeSH
- rozpouštědla chemie MeSH
- stabilita proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chitosan * MeSH
- kyselina polyglutamová * MeSH
- poly(gamma-glutamic acid) MeSH Prohlížeč
- rozpouštědla MeSH
Amyloidal protein fibrils occur in many biological events, but their formation and structural variability are understood rather poorly. We systematically explore fibril polymorphism for polyglutamic acid (PGA), insulin and hen egg white lysozyme. The fibrils were grown in the presence of "seeds", that is fibrils of the same or different protein. The seeds in concentrations higher than about 5 % of the total protein amount fully determined the structure of the final fibrils. Fibril structure was monitored by vibrational circular dichroism (VCD) spectroscopy and other techniques. The VCD shapes significantly differ for different fibril samples. Infrared (IR) and VCD spectra of PGA were also simulated using density functional theory (DFT) and a periodic model. The simulation provides excellent basis for data interpretation and reveals that the spectral shapes and signs depend both on fibril length and twist. The understanding of fibril formation and interactions may facilitate medical treatment of protein misfolding diseases in the future.
- Klíčová slova
- catalytic seeding, fibrous proteins, molecular modeling, protein folding, vibrational circular dichroism,
- MeSH
- amyloid analýza chemická syntéza MeSH
- cirkulární dichroismus MeSH
- inzulin chemie MeSH
- katalýza MeSH
- konformace proteinů MeSH
- kyselina polyglutamová chemie MeSH
- muramidasa chemie metabolismus MeSH
- teorie funkcionálu hustoty MeSH
- vibrace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- amyloid MeSH
- hen egg lysozyme MeSH Prohlížeč
- inzulin MeSH
- kyselina polyglutamová MeSH
- muramidasa MeSH
The 5-year mortality rate for heart failure borders on 50%. The main cause is an ischaemic cardiac event where blood supply to the tissue is lost and cell death occurs. Over time, this damage spreads and the heart is no longer able to pump efficiently. Increasing vascularisation of the affected area has been shown to reduce patient symptoms. The growth factors required to do this have short half-lives making development of an efficacious therapy difficult. Herein, the angiogenic growth factor Vascular Endothelial Growth Factor (VEGF) is complexed electrostatically with star-shaped or linear polyglutamic acid (PGA) polypeptides. Optimised PGA-VEGF nanomedicines provide VEGF encapsulation of > 99% and facilitate sustained release of VEGF for up to 28 days in vitro. The star-PGA-VEGF nanomedicines are loaded into a percutaneous delivery compliant hyaluronic acid hydrogel. Sustained release of VEGF from the composite nano-in-gel system is evident for up to 35 days and the released VEGF has comparable bioactivity to free, fresh VEGF when tested on both Matrigel® and scratch assays. The final star-PGA-VEGF nanomedicine-loaded hydrogel is biocompatible and provides sustained release of bioactive VEGF. Therefore, we report the development of novel, self-assembling PGA-VEGF nanomedicines and their incorporation into a hyaluronic acid hydrogel that is compatible with medical devices to enable minimally invasive delivery to the heart. The final star-PGA-VEGF nanomedicine-loaded hydrogel is biocompatible and provides sustained release of bioactive VEGF. This formulation provides the basis for optimal spatiotemporal delivery of an angiogenic growth factor to the ischaemic myocardium.
- Klíčová slova
- Angiogenesis, Growth factor, Ischaemia, Nanoparticle, Star polypeptide,
- MeSH
- aplikace kožní MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- hydrogely MeSH
- ischemická choroba srdeční farmakoterapie MeSH
- kyselina polyglutamová chemie MeSH
- léky s prodlouženým účinkem MeSH
- lidé MeSH
- nanočástice MeSH
- statická elektřina MeSH
- vaskulární endoteliální růstový faktor A chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hydrogely MeSH
- kyselina polyglutamová MeSH
- léky s prodlouženým účinkem MeSH
- vaskulární endoteliální růstový faktor A MeSH
In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.
- Klíčová slova
- H475Y(1561C→T) polymorphism, arene-binding site, crystal structure, folate hydrolase 1, zinc metalloprotease,
- MeSH
- antigeny povrchové chemie genetika MeSH
- glutamátkarboxypeptidasa II chemie genetika MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kyselina polyglutamová chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- polymorfismus genetický MeSH
- stabilita enzymů MeSH
- vazebná místa genetika MeSH
- vysoká teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- antigeny povrchové MeSH
- FOLH1 protein, human MeSH Prohlížeč
- folyl-n-gamma-L-glutamic acid MeSH Prohlížeč
- glutamátkarboxypeptidasa II MeSH
- kyselina polyglutamová MeSH
Highly ordered assemblies of β-sheet-forming peptide and protein fibrils have been the focus of much attention because of their multiple and partially unknown biological functions, in particular as related to degenerative neuronal disorders. Recently, vibrational circular dichroism (VCD) spectra have been shown to provide a unique means of detection for such extended structures utilizing modes of the peptide main chain backbone. In the case of poly-glutamic acid, surprising VCD responses were also found for side chain modes. In this study, in an attempt to explain this latter observation and obtain a link between fibrillar structure and its optical spectral properties, molecular dynamics (MD) methods are used to model the geometry and dynamics of assemblies containing repeating β-strands of Glu(n). A crystal-like model was adopted for the MD structure simulations. Infrared and VCD spectra for segments of MD modeled fibrillar geometries were first calculated using density functional theory (DFT), and then, those parameters were applied to larger structures by means of Cartesian coordinate transfer (CCT) of atomic tensors from the segments. The computations suggest the side chains exhibit residual conformational constraints, resulting in local coupling giving rise to non-negligible VCD intensity, albeit with an overall broad distribution. Calculated spectral distributions are qualitatively consistent with the experimental results but do differ in magnitude. The possibility of realistic modeling of vibrational spectra significantly broadens the potential for application of optical spectroscopies in structural studies of these aggregated biopolymers.
Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with gamma-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25-400 nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis.
- MeSH
- antirevmatika krev farmakokinetika terapeutické užití MeSH
- biologická dostupnost MeSH
- biotransformace MeSH
- erytrocyty metabolismus MeSH
- fluorescence MeSH
- hydrolýza MeSH
- juvenilní artritida krev farmakoterapie MeSH
- kalibrace MeSH
- kyselina polyglutamová analogy a deriváty krev farmakokinetika MeSH
- lidé MeSH
- methotrexát analogy a deriváty krev farmakokinetika terapeutické užití MeSH
- monitorování léčiv metody normy MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie * normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antirevmatika MeSH
- kyselina polyglutamová MeSH
- methotrexát MeSH
- methotrexate polyglutamate MeSH Prohlížeč
OBJECTIVE: We assessed the effect of folic acid (FA) on the pharmacokinetics and pharmacodynamics of low-dose oral methotrexate (MTX) during the remission-induction phase of psoriasis treatment. METHODS: In a 32-week, open-label, two-way cross-over study, patients (n=20, seven men, aged 35-70 years) with moderate-to-severe plaque psoriasis were randomly assigned to receive MTX plus FA (20 mg/week) for 16 weeks followed by MTX monotherapy (three doses of MTX separated by 12-h intervals once a week) for an additional 16 weeks (treatment arm A, n=10) or to receive the opposite sequence of treatments (arm B, n=10). Dosing of MTX was individualised with the help of pre-study evaluation of plasma MTX pharmacokinetics. The Psoriasis Area and Severity Index (PASI), biochemistry and haematology tests and erythrocyte concentration of MTX polyglutamates (MTXPG) were evaluated throughout the study. RESULTS: In arms A and B, the mean (range) concentrations of MTXPG (nmol/L) were comparable [week 16: 96.2 (32.0-157) vs. 111 (73.7-175), P=0.32; week 32: 103 (55.8-173) vs. 83.6 (27.4-129), P=0.24]. After 16 weeks, the mean+/-SEM PASI decreased from 20.1+/-2.1 to 8.8+/-1.3 in arm A, while a greater reduction from 27.2+/-2.1 to 5.1+/-1.0 occurred in arm B (P<0.001). Positive correlations were found between the percent improvement in PASI at week 16 and the ratios of the concentration of MTXPG to plasma folate (rho=0.59, P=0.008) or RBC folate concentration (rho=0.56, P=0.013). Due to an accelerated decline in PASI in arm A and a trend to its worsening in arm B after crossing over of treatments, the mean absolute PASI scores in both arms were comparable at week 32. CONCLUSION: The antipsoriatic effect of MTX during the remission-induction phase of treatment is influenced by folate status and may be significantly less if combined treatment with FA is used, irrespective of pre-treatment folate levels. The individual tailoring of MTX dosing needs further attention because the mean percent PASI improvement from baseline was 83% and the inter-patient variability in response was low after 16 weeks of monotherapy with MTX.
- MeSH
- adherence pacienta MeSH
- antagonisté kyseliny listové škodlivé účinky farmakokinetika terapeutické užití MeSH
- aplikace orální MeSH
- dermatologické látky škodlivé účinky farmakokinetika terapeutické užití MeSH
- erytrocyty metabolismus MeSH
- klinické křížové studie MeSH
- kyselina listová škodlivé účinky krev terapeutické užití MeSH
- kyselina polyglutamová krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- methotrexát škodlivé účinky farmakokinetika terapeutické užití MeSH
- potravní doplňky MeSH
- psoriáza farmakoterapie MeSH
- senioři MeSH
- vitaminy škodlivé účinky krev terapeutické užití MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- randomizované kontrolované studie MeSH
- Názvy látek
- antagonisté kyseliny listové MeSH
- dermatologické látky MeSH
- kyselina listová MeSH
- kyselina polyglutamová MeSH
- methotrexát MeSH
- vitaminy MeSH
The interactions of poly-L-glutamic acid and a cationic porphyrin derivative in aqueous solutions were studied by the combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies. It was found that non-covalent interactions between both agents influence the structure of the polymeric matrix and the guest porphyrins and vice versa, but the physico-chemical properties of the solutions, especially the pH and the relative permittivity of the solvent, play a key role in the structure of the polypeptide part of the formed complexes. It was shown that the interaction with porphyrins prevents the precipitation of poly-L-glutamic acid in aqueous solution at acidic pH. In special conditions, the porphyrins attached to the polypeptide probably possess face-to-face interaction as demonstrated by the enhancement of the characteristic ECD signal and the appearance of sidebands on its short and long wavelength sides.
- MeSH
- cirkulární dichroismus MeSH
- kyselina polyglutamová chemie MeSH
- molekulární struktura MeSH
- porfyriny chemie MeSH
- roztoky MeSH
- spektrofotometrie ultrafialová MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyselina polyglutamová MeSH
- porfyriny MeSH
- roztoky MeSH
- voda MeSH
