Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 ...interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNA
-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNA
was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.
The parasites Trypanosoma brucei (Tb) and Leishmania major (Lm) cause the tropical diseases sleeping sickness, nagana, and cutaneous leishmaniasis. Every year, millions of humans, as well as animals, ...living in tropical to subtropical climates fall victim to these illnesses’ health threats. The parasites’ frequent drug resistance and widely spread natural reservoirs heavily impede disease prevention and treatment. Due to pteridine auxotrophy, trypanosomatid parasites have developed a peculiar enzyme system consisting of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) to support cell survival. Extending our previous studies, we conducted a comparative study of the T. brucei (TbDHFR, TbPTR1) and L. major (LmDHFR, LmPTR1) enzymes to identify lead structures with a dual inhibitory effect. A pharmacophore-based in silico screening of three natural product databases (approximately 4880 compounds) was performed to preselect possible inhibitors. Building on the in silico results, the inhibitory potential of promising compounds was verified in vitro against the recombinant DHFR and PTR1 of both parasites using spectrophotometric enzyme assays. Twelve compounds were identified as dual inhibitors against the Tb enzymes (0.2 μM < IC50 < 85.1 μM) and ten against the respective Lm enzymes (0.6 μM < IC50 < 84.5 μM). These highly promising results may represent the starting point for the future development of new leads and drugs utilizing the trypanosomatid pteridine metabolism as a target.
Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an ...enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.
The enzymatic degradation of polyethylene terephthalate (PET) is a promising new approach for the environmentally friendly recycling of PET waste, but so far, low degradation rates paired with ...releatively high costs have limited the economic feasibility of this method. Here we present the construction of a new bacterial whole‐cell biocatalyst utilizing a new inverse autotranspoter based surface display of PETase in E. coli. The resulting catalyst allows an extremely easy production of large amounts of enzyme and a five times more effective degradation of PET at a lower temperature of 25 °C compared to free PETase at 30 °C. Additionally, we demonstrate how rhamnolipids, which are environmentally benign and can be cost‐effectively produced using strains of E. coli, can be used to amplify the hydrolysis rates of PET even further, presumably by acting as mediators between PETase and PET. Cells displaying PETase in combination with externally supplied rhamnolipids outperformed free PETase by a factor of 16, allowing the degradation of highly crystalline post‐consumer PET waste to an extent of 8 % within 3 days at room temperature.
Whole‐cell biocatalyst: A new E. coli whole‐cell biocatalyst with surface displayed PETase can efficiently degrade post‐consumer PET at room temperature into its constituents, which could be further used to generate value‐added products from waste. The addition of rhamnolipids as environmentally benign biosurfactants further increases the extent of degradation.
The parasite
(
.
) is responsible for human African trypanosomiasis (HAT) and the cattle disease "Nagana" which to this day cause severe medical and socio-economic issues for the affected areas in ...Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (
DHFR-TS) and the pteridine reductase 1 (
PTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the
PTR1 and
DHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against
PTR1 as well as three compounds against
DHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both
PTR1 and
DHFR, reaching IC
values of 12.4 µM and 7.1 µM, respectively.
Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are promising therapeutic targets because of their association with the genesis of several diseases. The identification of ...selective compounds that alter cAMP-induced ion channel modulation by binding to the cyclic nucleotide-binding domain (CNBD) will facilitate HCN channel-specific drug development. In this study, a fast and protein purification-free ligand-binding approach with a surface-displayed HCN4 C-Linker-CNBD on
is presented. 8-Fluo-cAMP ligand binding was monitored by single-cell analysis via flow cytometry, and a K
-value of 173 ± 46 nM was determined. The K
value was confirmed by ligand depletion analysis and equilibrium state measurements. Applying increasing concentrations of cAMP led to a concentration-dependent decrease in fluorescence intensity, indicating a displacement of 8-Fluo-cAMP. A K
-value of 8.5 ± 2 µM was determined. The linear relationship of IC
values obtained for cAMP as a function of ligand concentration confirmed the competitive binding mode: IC
: 13 ± 2 µM/16 ± 3 µM/23 ± 1 µM/27 ± 1 µM for 50 nM/150 nM/250 nM/500 nM 8-Fluo-cAMP. A similar competitive mode of binding was confirmed for 7-CH-cAMP, and an IC
value of 230 ± 41 nM and a K
of 159 ± 29 nM were determined. Two established drugs were tested in the assay. Ivabradine, an approved HCN channel pore blocker and gabapentin, is known to bind to HCN4 channels in preference to other isoforms with an unknown mode of action. As expected, ivabradine had no impact on ligand binding. In addition, gabapentin had no influence on 8-Fluo-cAMP's binding to HCN4-CNBD. This is the first indication that gabapentin is not interacting with this part of the HCN4 channel. The ligand-binding assay as described can be used to determine binding constants for ligands such as cAMP and derivatives. It could also be applied for the identification of new ligands binding to the HCN4-CNBD.
A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation ...control, antibody-binding Z-domain-autodisplaying
(
) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)
), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The
-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of
cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.
Being an essential multifunctional platform and interface to the extracellular environment, the cell membrane constitutes a valuable target for the modification and manipulation of cells and cellular ...behavior, as well as for the implementation of artificial, new-to-nature functionality. While bacterial cell surface functionalization via expression and presentation of recombinant proteins has extensively been applied, the corresponding application of functionalizable lipid mimetics has only rarely been reported. Herein, we describe an approach to equip E. coli cells with a lipid-mimicking, readily membrane-integrating imidazolium salt and a corresponding NHC–palladium complex that allows for flexible bacterial membrane surface functionalization and enables E. coli cells to perform cleavage of propargyl ethers present in the surrounding cell medium. We show that this approach can be combined with already established on-surface functionalization, such as bacterial surface display of enzymes, i.e. laccases, leading to a new type of cascade reaction. Overall, we envision the herein presented proof-of-concept studies to lay the foundation for a multifunctional toolbox that allows flexible and broadly applicable functionalization of bacterial membranes.
To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and ...biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 10⁵ recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.
Heat shock protein 90 (HSP90) is a critical target for anticancer and anti-fungal-infection therapies due to its central role as a molecular chaperone involved in protein folding and activation. In ...this study, we developed
Förster Resonance Energy Transfer (FRET) assays to characterize the binding of
HSP90 to its co-chaperone Sba1, as well as that of the homologous
HSP90α to p23. The assay for
HSP90α binding to p23 enables selectivity assessment for compounds aimed to inhibit the binding of
HSP90 to Sba1 without affecting the physiological activity of
HSP90α. The combination of the two assays is important for antifungal drug development, while the assay for
HSP90α can potentially be used on its own for anticancer drug discovery. Since ATP binding of HSP90 is a prerequisite for HSP90-Sba1/p23 binding, ATP-competitive inhibitors can be identified with the assays. The specificity of binding of fusion protein constructs-HSP90-mNeonGreen (donor) and Sba1-mScarlet-I (acceptor)-to each other in our assay was confirmed via competitive inhibition by both non-labeled Sba1 and known ATP-competitive inhibitors. We utilized the developed assays to characterize the stability of both HSP90-Sba1 and HSP90α-p23 affinity complexes quantitatively.
values were determined and assessed for their precision and accuracy using the 95.5% confidence level. For HSP90-Sba1, the precision confidence interval (PCI) was found to be 70-120 (100 ± 20) nM while the accuracy confidence interval (ACI) was 100-130 nM. For HSP90α-p23, PCI was 180-260 (220 ± 40) nM and ACI was 200-270 nM. The developed assays were used to screen a nucleoside-mimetics library of 320 compounds for inhibitory activity against both
HSP90-Sba1 and
HSP90α-p23 binding. No novel active compounds were identified. Overall, the developed assays exhibited low data variability and robust signal separation, achieving
factors > 0.5.
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