Refine
Document Type
- Article (17)
Language
- English (17)
Has Fulltext
- yes (17)
Is part of the Bibliography
- no (17)
Keywords
- - (5)
- BCL11B (2)
- crystal structure (2)
- homodimerization (2)
- pancreas (2)
- pancreatitis (2)
- serine protease (2)
- CCHC zinc finger (1)
- FRET (1)
- Kazal inhibitor (1)
- Molecular dynamics simulation (1)
- N34S (1)
- PHMB (1)
- Poly(hexamethylene)biguanide (1)
- R122H (1)
- Rose Bengal staining (1)
- TIGER2h (1)
- TIGER2hs (1)
- Ti6Al4V (1)
- TiO2 (1)
- antimicrobial coating (1)
- anti‐PF4/heparin antibodies (1)
- autoproteolysis (1)
- carbon contamination (1)
- catalytic triad (1)
- detergents (1)
- implant coating (1)
- integrin αIIbβ3 (1)
- interaction (1)
- isothermal titration calorimetry (ITC) (1)
- length (1)
- lipids (1)
- liposomes (1)
- micelle (1)
- molecular dynamics simulations (MDS) (1)
- osseointegration (1)
- platelet factor 4 (1)
- protein expression and purification (1)
- protein folding (1)
- protein-protein docking (1)
- protein–protein interaction (1)
- reconstitution (1)
- replica-exchange molecular dynamics (1)
- standard mechanism (1)
- surface plasmon resonance (SPR) (1)
- synthetic heparins (1)
- water contact angle (1)
- wetting (1)
- zinc finger (1)
Institute
Publisher
- MDPI (5)
- Wiley (2)
- Dove Medical Press (1)
- Nature Publishing Group (1)
- Springer Nature (1)
Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.
(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1–TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.
Objective: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). This ratio is reversed during chronic alcohol abuse, pancreatic cancer, or pancreatitis due to selectively upregulated expression of TRY2, causing anionic trypsin to become the predominant isoform. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity.
Methods: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model.
Results: All trypsin isoforms display similar kinetic properties. The crystal structure of TRY2 reveals that the enzyme crystallized in the autoproteolytic state with Arg122 placed in the S1 binding pocket and the corresponding loop cleaved. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface.
Conclusion: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated.
Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.
Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.
Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations
(2021)
Transcription factors play a crucial role in regulating biological processes such as cell
growth, differentiation, organ development and cellular signaling. Within this group, proteins
equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding
transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a
variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary
glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which
serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown
to be critically important for efficient regulation of the BCL11B target genes and could therefore
represent a promising target for novel drug therapies. Here, we report the structural basis for
BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure
prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy
transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of
the single ZF domain and directly involved in forming the dimer interface. These findings not only
provide deep insight into how BCL11B acquires its active structure but also represent an important
step towards rational design or selection of potential inhibitors.
Unlike the native surface of the implant material (Ti6Al4V), oxidation with H2O2 leads to increased binding of the effective antimicrobial agent poly(hexamethylene) biguanide [PHMB]. However, treating with NaOH instead results in an even higher PHMB mass coverage. After oxidation with H2O2, strong differences in the PHMB adsorption capability between polished and corundum-blasted surfaces appear, indicating a roughness dependence. After NaOH treatment, no such effect was observed. The wetting properties of specimens treated with either H2O2 or NaOH prior to PHMB exposure clearly varied. To unravel the nature of this interaction, widespread in silico and in vitro experiments were performed. Methods: By X-ray photoelectron spectroscopy, scanning electron microscopy, water contact angle measurements and MD simulations, we characterized the interplay between the polycationic antimicrobial agent and the implant surface. A theoretical model for PHMB micelles is tested for its wetting properties and compared to carbon contaminated TiO2. In addition, quantitation of anionic functional group equivalents, the binding properties of PHMB with blocked amino end-group, and the ability to bind chlorhexidine digluconate (CHG) were investigated. Ultimately, the capability of osteoblasts to build calcium apatite, and the activity of alkaline phosphatase on PHMB coated specimens, were determined. Results: Simulated water contact angles on carbon contaminated TiO2 surfaces and PHMB micelle models reveal little influence of PHMB on the wetting properties and point out the major influence of remaining and recovering contamination from ambient air. Testing PHMB adsorption beyond the critical micelle concentration and subsequent staining reveals an island-like pattern with H2O2 as compared to an evenly modified surface with NaOH. Both CHG and PHMB, with blocked amino end groups, were adsorbed on the treated surfaces, thus negating the significant influence of PHMB’s terminal groups. The ability of osteoblasts to produce calcium apatite and alkaline phosphatase is not negatively impaired for PHMB mass coverages up to 8 μg/specimen. Conclusion: Differences in PHMB adsorption are triggered by the number of anionic groups and carbon contaminants, both of which depend on the specimen pre-treatment. With more PHMB covering, the implant surface is protected against the capture of new contamination from the ambient air, thus building a robust antimicrobial and biocompatible surface coating.
Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder
antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and
comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide
bond at domain V has been associated with different cysteine redox states. The role of this disulfide
bond in conformational dynamics of this protein has not been investigated so far. Here, we report
on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine;
TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry
analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images
suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more
flexible compared to the untreated protein as confirmed by modelling studies. We have determined a
strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated
by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein
structure and its implications for antibody binding in APS patients.
Abstract
Background
Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin‐induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6‐, 8‐, 10‐, and 12‐mer size and a hypersulfated 12‐mer (S12‐mer).
Methods
We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti‐PF4/heparin antibodies.
Results
The synthetic heparin‐like compounds display stronger binding characteristics to PF4 than animal‐derived heparins of corresponding lengths. Upon complexation with PF4, 6‐mer and S12‐mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8‐, 10‐, and 12‐mer heparins. Anti‐PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ≤ 8‐mer than with complexes formed between PF4 and heparins ≥ 10‐mer. Addition of one sulfate group to the 12‐mer resulted in a S12‐mer, which showed substantial changes in its binding characteristics to PF4.
Conclusions
We provide a template for characterizing interactions of newly developed heparin‐based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.