9 research outputs found
Asymmetric DNA-Search Dynamics by Symmetric Dimeric Proteins
We focus on dimeric DNA-binding proteins
from two well-studied
families: orthodox type II restriction endonucleases (REs) and transcription
factors (TFs). Interactions of the protein’s recognition sites
with the DNA and, particularly, the contribution of each of the monomers
to one-dimensional (1D) sliding along nonspecific DNA were studied
using computational tools. Coarse-grained molecular dynamics simulations
of DNA scanning by various TFs and REs provide insights into how the
symmetry of a homodimer can be broken while they nonspecifically interact
with DNA. The characteristics of protein sliding along DNA, such as
the average sliding length, partitioning between 1D and 3D search,
and the one-dimensional diffusion coefficient <i>D</i><sub>1</sub>, strongly depend on the salt concentration, which in turn
affects the probability of the two monomers adopting a cooperative
symmetric sliding mechanism. Indeed, we demonstrate that maximal DNA
search efficiency is achieved when the protein adopts an asymmetric
search mode in which one monomer slides while its partner hops. We
find that proteins classified as TFs have a higher affinity for the
DNA, longer sliding lengths, and an increased probability of symmetric
sliding in comparison with REs. Moreover, TFs can perform their biological
function over a much wider range of salt concentrations than REs.
Our results demonstrate that the different biological functions of
DNA-binding proteins are related to the different nonspecific DNA
search mechanisms they adopt
Inhibition of AcrAB-TolC enhances antimicrobial activity of phytochemicals in Pectobacterium brasiliense
IntroductionThe eons-long co-evolvement of plants and bacteria led to a plethora of interactions between the two kingdoms, in which bacterial pathogenicity is counteracted by plant-derived antimicrobial defense molecules. In return, efflux pumps (EP) form part of the resistance mechanism employed by bacteria to permit their survival in this hostile chemical environment. In this work we study the effect of combinations of efflux pump inhibitors (EPIs) and plant-derived phytochemicals on bacterial activity using Pectobacteriun brasiliense 1692 (Pb1692) as a model system.MethodsWe measured the minimal inhibitory concentration (MIC) of two phytochemicals, phloretin (Pht) and naringenin (Nar), and of one common antibiotic ciprofloxacin (Cip), either alone or in combinations with two known inhibitors of the AcrB EP of Escherichia coli, a close homolog of the AcrAB-TolC EP of Pb1692. In addition, we also measured the expression of genes encoding for the EP, under similar conditions.ResultsUsing the FICI equation, we observed synergism between the EPIs and the phytochemicals, but not between the EPIs and the antibiotic, suggesting that EP inhibition potentiated the antimicrobial activity of the plant derived compounds, but not of Cip. Docking simulations were successfully used to rationalize these experimental results.DiscussionOur findings suggest that AcrAB-TolC plays an important role in survival and fitness of Pb1692 in the plant environment and that its inhibition is a viable strategy for controlling bacterial pathogenicity
Design of Compact Biomimetic Cellulose Binding Peptides as Carriers for Cellulose Catalytic Degradation
The
conversion of biomass into biofuels can reduce the strategic
vulnerability of petroleum-based systems and at the same time have
a positive effect on global climate issues. Lignocellulose is the
cheapest and most abundant source of biomass and consequently has
been widely considered as a source for liquid fuel. However, despite
ongoing efforts, cellulosic biofuels are still far from commercial
realization, one of the major bottlenecks being the hydrolysis of
cellulose into simpler sugars. Inspired by the structural and functional
modularity of cellulases used by many organisms for the breakdown
of cellulose, we propose to mimic the cellulose binding domain (CBD)
and the catalytic domain of these proteins by small molecular entities.
Multiple copies of these mimics could subsequently be tethered together
to enhance hydrolytic activity. In this work, we take the first step
toward achieving this goal by applying computational approaches to
the design of efficient, cost-effective mimetics of the CBD. The design
is based on low molecular weight peptides that are amenable to large-scale
production. We provide an optimized design of four short (i.e., ∼18
residues) peptide mimetics based on the three-dimensional structure
of a known CBD and demonstrate that some of these peptides bind cellulose
as well as or better than the full CBD. The structures of these peptides
were studied by circular dichroism and their interactions with cellulose
by solid phase NMR. Finally, we present a computational strategy for
predicting CBD/peptide–cellulose binding free energies and
demonstrate its ability to provide values in good agreement with experimental
data. Using this computational model, we have also studied the dissociation
pathway of the CBDs/peptides from the surface of cellulose
Structure–Activity Relationship of Synthetic Linear KTS-Peptides Containing Meta-Aminobenzoic Acid as Antagonists of α1β1 Integrin with Anti-Angiogenic and Melanoma Anti-Tumor Activities
To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed
with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins,
which selectively block collagen α1β1 integrin, were used as lead compounds for the synthesis
and structure–activity relationship of a series of linear peptides containing the KTS-pharmacophore
and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness
and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore
motif. Molecular dynamics simulations determined that the solution conformation of
MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and
spent most of the time in a single cluster. The peptides’ binding affinity has been characterized by
an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50
of 324 ± 8 μM and 550 ± 45 μM the binding of GST-α1-A domain to collagen IV fragment CB3,
and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies
and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near
the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited
tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide
4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and
angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells.
These findings propose that MABA-peptide 4 can further serve as an α1β1-integrin antagonist lead
compound for further drug optimization in angiogenesis and cancer therapy
A novel image-based high-throughput screening assay discovers therapeutic candidates for adult polyglucosan body disease
Glycogen storage disorders (GSDs) are caused by excessive accumulation of glycogen. Some GSDs (Adult Polyglucosan Body Disease (APBD), Tarui and Lafora diseases) are caused by intracellular accumulation of insoluble inclusions, called polyglucosan bodies (PB), which are chiefly composed of malconstructed glycogen. We developed an APBD patient skin fibroblast cell-based assay for PB identification, where the bodies are identified as amylase-resistant periodic acid-Schiff's (PAS) stained structures, and quantified. We screened the DIVERSet-CL 10,084 compound library using this assay in high throughput format and discovered 11 dose-dependent and 8 non dose-dependent PB-reducing hits. ~70% of the hits appear to act through reducing glycogen synthase (GS) activity which can elongate glycogen chains and presumably promote PB generation. Some of these GS inhibiting hits were also computationally predicted to be similar to drugs interacting with the GS activator protein phosphatase 1 (PP1). Our work paves the way to discovering medications for the treatment of PB-involving GSD, which are extremely severe or fatal disorders