Biology subjects

Athanasiou, C.

Publications and source records attributed to Athanasiou, C..

5 recordsLinked to original sources

bioRxiv2026

A metagenomic thermostable monomeric meganuclease with novel specificity and unique palindromic 3-prime overhangs

As an alternative to historical enzyme isolation, metagenomic databases (e.g. MGnify) provide information on vast unculturable microbial diversity, especially from extreme environments, and constitute an enormous source of functional proteins. Conservative mining of these data by close sequence homology alone tends to identify merely different versions of known enzymes. Here we present a discovery strategy of meganucleases based on wider capture of less homologous enzymes with new function in metagenomic databases, incorporating metadata with homology, relying on cell-free expression to bypass host incompatibility and the need for purification, along with using deep sequencing for experimental assessment of substrate specificity and cleavage pattern, circumventing classical gel-based profiling. Specifically, we discovered the temperature-stable (>55{degrees}C), intron-encoded LAGLIDADG meganuclease I-MG11 that recognizes a 17 base pair sequence to generate unique 4 base pair palindromic 3'-overhangs -- the first monomeric meganuclease to produce such overhangs. Co-folding models of I-MG11 bound to DNA provide a structural context for enzyme-DNA interactions, highlighting differences from other monomeric LAGLIDADG meganucleases (e.g. I-SceI) shaped by InDels (insertion-deletions) in the DNA binding region that may cause specificity changes. Our strategy streamlines bona fide identification and annotation of meganucleases, while the unique properties of I-MG11 expand the molecular biology toolbox. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=31 SRC="FIGDIR/small/712669v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@199394aorg.highwire.dtl.DTLVardef@806490org.highwire.dtl.DTLVardef@14a533aorg.highwire.dtl.DTLVardef@9e13db_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Glycosylation-Modulated Conformational Diversity in Neurotrophin Receptors

Glycosylation is a widespread modification of cell-surface receptors, yet its structural impact is often overlooked due to the difficulty of experimentally characterizing glycans. Glycosylation of neurotrophin receptors has been reported to affect their localization and function. To investigate the effects of glycosylation of the extracellular domains (ECDs) of p75, TrkA, and TrkB neurotrophin receptors, we modeled their ECDs in glycosylated and non-glycosylated states and carried out molecular dynamics simulations of monomeric and dimeric forms of the ECDs with and without a neurotrophin bound. The single N-glycan on the p75 ECD provided minimal shielding and had limited interaction with the neurotrophin, although glycan-glycan contacts between the two p75 monomers may influence the stability of the receptor-neurotrophin complex. In contrast, TrkA and TrkB carry multiple N-glycans that shield the ECDs much more and, particularly for TrkB, increase the contact area between the receptor and the neurotrophin. The p75 ECD was comparatively rigid, independent of glycosylation state, likely due to its extensive network of disulfide bonds. In contrast, without glycans, the TrkA and TrkB ECDs tended to collapse inwards, sometimes obstructing the neurotrophin binding site. Glycosylation of TrkA and TrkB prevented bending of the ECD into more compact states, and instead promoted extended conformations that better accommodate neurotrophin binding. Overall, the simulations reveal distinct, receptor-specific roles of glycosylation in modulating neurotrophin receptor shielding, flexibility, and conformation, with effects on neurotrophin binding. Statement of significanceNeurotrophins are proteins that promote neuronal survival. Their binding to the extracellular domains of transmembrane receptors leads to signal transduction intracellularly. Glycosylation of some neurotrophin receptors has been reported to affect their localization and function but the mechanism is unclear. We investigate the effects of glycosylation on the structure, dynamics and binding properties of the extracellular domains of three neurotrophin receptors by molecular dynamics simulation. We identify distinct receptor-specific effects of glycosylation. In particular, we find that glycosylation of tyrosine receptor kinases prevents concealment of the neurotrophin binding site due to receptor dynamics and favors a more extended receptor conformation, thereby supporting neutrophin binding and the associated signal transduction.

biophysics↗

bioRxiv2024

Simulation of neurotrophin receptor transmembrane helix interactions reveals active states and distinct signaling mechanisms

Neurotrophin (NT) receptor signaling regulates neuronal survival, axonal and dendritic network maintenance, differentiation, and synaptic plasticity. Signaling is initiated by NT binding to the extracellular domain of NT receptor dimers, leading to activation of the receptor and signal propagation intracellularly. How this activating signal is mediated by the single-pass transmembrane (TM) helical domain of the receptor, and what the relation between domain sequence and signaling mechanism is, remains unclear. The structure and dynamics of the TM domain of the receptor dimers in the active and inactive states for intracellular signaling are still elusive, with NMR structures capturing only a single state. Here, we carried out unbiased and enhanced sampling molecular dynamics simulations of the TM domain dimers of the wild-type p75, TrkA and TrkB NT receptors and selected mutants in micelle and bilayer lipid environments at atomistic and coarse-grained levels of representation. The coarse-grained simulations enabled exploration of multiple states of the TM domain dimers and revealed the influence of the lipid environment on the TM helix arrangements. From the simulations, we identify active and inactive TM helix arrangements of the p75 and TrkA receptors that are supported by experimental data and suggest two different signaling mechanisms through the C-terminal regions of the TM helices. For TrkB, a single dominant but less energetically stable arrangement of the TM domain dimer is observed. These findings have implications for mechanistic studies of NT receptor signaling and the design of neuroprotective drugs to stabilize specific states of the TM domain of the receptors. Significance StatementNeurotrophins regulate neuronal survival, growth and differentiation during development, and play a role in many neurodegenerative and psychiatric disorders. They initiate signaling through the cell membrane by associating extracellularly with transmembrane receptors. The transmembrane helical domain of the neurotrophin receptors is responsible for transmitting the activation signal to the cell interior. However, how the transmembrane domain mediates signal transmission and the relation between its sequence and signaling mechanism remain unclear. Here, by employing state-of-the-art molecular dynamics simulation techniques, we identify active and inactive states of the transmembrane domains of the three main neurotrophin receptors that support distinct transmembrane signaling mechanisms for these receptors. Our results have implications for mechanistic studies of neurotrophin signaling and the design of neuroprotective drugs.

molecular biology↗

bioRxiv2023

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer's Disease

Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimers Disease (AD) pathology. Addressing these impairments is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. In our study, we use a combination of mouse and human stem cell models to circumvent these issues and perform comprehensive characterization of a novel neurogenic compound using in vitro screening. Our work focuses on the brain-derived neurotrophic factor (BDNF) pathway, a pivotal neurotrophin in the regulation of neuronal growth and differentiation via its receptor tyrosine receptor kinase B (TrkB). We describe the design, chemical synthesis and biological characterization of ENT-A011, a steroidal dehydroepiandrosterone (DHEA) derivative and BDNF mimetic with neuroprotective and neurogenic actions. The compound is able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Amyloid-{beta} (A{beta}) induced cell death, acting specifically through TrkB activation. The compound is also able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from A{beta} toxicity and prevent synapse loss after A{beta} treatment. To provide a translational link to human cells, we also used neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. Our findings suggest that ENT-A011 successfully induces proliferation and prevents cell death after A{beta} toxicity of human NPCs. Additionally, using RNAseq profiling, we demonstrate that the compound acts through a core gene network shared with BDNF. Our work characterizes a novel synthetic BDNF mimetic with potential neurogenic and neuroprotective actions in Alzheimers disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

pharmacology and toxicology↗

bioRxiv2022

Scaling protein-water interactions in the Martini 3 coarse-grained force field to simulate transmembrane helix dimers in different lipid environments

Martini 3, the latest version of the widely used Martini force field for coarse-grained molecular dynamics simulations, is a promising tool to investigate proteins in phospholipid bilayers. However, simulating other lipid environments, such as detergent micelles, presents challenges due to the absence of validated parameters for their constituent molecules. Here, we propose parameters for the micelle-forming surfactant, dodecylphosphocholine (DPC). These result in micelle assembly with aggregation numbers in agreement with experimental values. However, we identified a lack of hydrophobic interactions between transmembrane helix protein dimers and the tails of DPC molecules, preventing insertion and stabilization of the protein in the micelles. This problem was also observed for protein insertion by self-assembling 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or dipalmitoylphosphatidylcholine (DPPC) bilayers. We propose the reduction of the non-bonded interactions between protein and water beads by 10% as a simple and effective solution to this problem that enables protein encapsulation in phospholipid micelles and bilayers without altering protein dimerization or bilayer structure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/506752v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@105d651org.highwire.dtl.DTLVardef@398758org.highwire.dtl.DTLVardef@e4c045org.highwire.dtl.DTLVardef@afdc1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗