Biology subjects

Ilie, I. M.

Publications and source records attributed to Ilie, I. M..

9 recordsLinked to original sources

bioRxiv2026

Physical mechanisms of nanoparticle intracellular release through membrane pore nucleation and expansion

Successful intracellular delivery requires a nanocarrier to adhere to the cell membrane, be wrapped and internalized and finally released into the cytoplasm before it is degraded. While the initial steps have been intensively studied, the intracellular release of a nanocarrier has received comparatively less attention and remains poorly understood. To understand the physical mechanisms that govern intracellular release, we investigate the escape of nanocarriers (radii 4.5{sigma} and 7{sigma} ) from a lipid vesicle using coarse-grained simulations. We represent a nanocarrier using the metaparticle model and the membrane using the three-beads Cooke-Deserno model. Starting from a fully endocytosed state, we systematically vary the repulsion between the nanoparticle and the lipid head groups and compare three release scenarios: passive release, localized inside-out activity from a drug-like source confined within the particle and activity distributed over the entire nanoparticle. An elastic-energy analysis shows that in a tensionless membrane escape requires crossing a large pore-expansion barrier of {approx} 68kBT for the smaller particle and {approx} 110kBT for the larger, making passive release thermodynamically improbable. Our results show that the smaller particle nonetheless escapes above a threshold repulsion, whereas the larger one remains trapped in a metastable state in which local membrane pores form but fail to grow beyond the particle diameter to allow successful release. Adding activity lowers the nucleation barrier and promotes earlier pore opening, but its effectiveness depends on how it is applied, i.e., a localized internal source assists release of the smaller particle yet is insufficient to systematically free the larger one, while activity distributed across the whole nanoparticle drives complete release even in the larger case. These results show that productive escape is governed by whether the resulting pore can evolve into a geometrically accessible release pathway, providing physical design principles for intracellular delivery by nanocarriers

biophysics↗

bioRxiv2026

Computationally engineered cyclic peptides reduce prion levels in vitro

Prion diseases are neurodegenerative disorders associated with the structural conversion of the cellular prion protein (PrPc) into its misfolded infectious isoform (PrPSc). Despite substantial efforts, no disease-modifying therapy or cure is currently available. Here, we present an integrated computational-experimental pipeline for the rational design of cyclic peptides targeting PrPc to inhibit its pathogenic conversion. Starting from crystal structures of antibody-bound mouse PrPc, we develop a rational design strategy combined with iterative molecular dynamics simulations and sequence optimization to generate peptides with enhanced binding and structural impact. Three candidates were selected for experimental validation. Our results show that [Formula] (49YGPDPSDSYT58, antibody numbering) that binds stably to the 2-3 interface most effectively reduced PrPSc levels in GT1-7 cells, essentially by inducing allosteric re-arrangements that reinforce the intramolecular helical bundle. [Formula] (89GQSNTKPYT97) and [Formula] (89RQSNTWPYT97) binding the {beta}1-1/3 junction exerted more modest effects due to the potential competition of the flexible tail to bind at this site. These results establish a mechanistic link between peptide-induced stabilization of PrPc and inhibition of prion propagation and provide a generalizable framework for designing conformational stabilizers of aggregation-prone proteins.

biophysics↗

bioRxiv2025

Physical mechanisms of nanoparticle-membrane interactions: A coarse-grained study

Nanoparticles (NPs) are promising drug carriers for targeted therapies, diagnostic imaging and advanced vaccines. However, their clinical translation is limited by complex biological barriers that reduce cellular uptake and efficacy. Specifically, the interaction with the cellular membrane controls nanoparticle adhesion, wrapping or full engulfment, which ultimately govern nanoparticle internalization efficiency. Flexible nanocarriers (e.g. liposomes, polymeric, micelles) are particularly attractive because their deformability could help them enhance the probability of successful cellular entry. To understand the physical mechanisms associated with cellular uptake, we investigate the interaction of semi-flexible nanocarriers with a symmetric lipid bilayer using coarse-grained simulations. We represent a flexible nanoparticle using the previously introduced MetaParticle (MP) model and the membrane using the Cooke-Deserno model. By systematically varying nanoparticle properties, i.e., adhesion strength and topology, we identify distinct interaction regimes ranging from surface adhesion and trapping to complete wrapping and endocytosis. These regimes correlate with nanoparticle shape, size and surface properties, providing quantitative design principles for optimizing cellular uptake. Overall, this framework offers predictive insight into how the interplay between nanoparticle properties and membrane interaction governs cellular internalization, informing the rational design of next-generation soft nanocarriers.

biophysics↗

bioRxiv2024

Computational design of Bax-inhibiting peptides

The proteins of the Bcl-2 family play crucial roles in regulating apoptosis. It is divided into pro-survival and pro-apoptotic proteins that determine cellular fate. In particular, Bax is a crucial executor of apoptosis as its activation initiates the apoptotic phenotype. Hence, targeting this protein represents an attractive therapeutic approach, which can aid in regulating apoptotic signalling and potentially contribute to the development of novel therapies against cancer and neurodegenerative diseases. Here, we introduce a digital paradigm, which relies on rational design and computer simulations to develop and validate peptide-based agents that bind to Bax, thereby inhibiting its apoptotic properties. The peptides are rationally designed and optimized to bind to Bax starting from the crystal structures of affimers in complex with Bcl-2 proteins. Next, molecular dynamics simulations (MD) are employed to probe the stability of the Bax-peptide complexes and to estimate the binding free energies. The results show that the designed peptides bind with high affinity to Bax. Two of the designed peptides bind in the canonical hydrophobic groove (BH1 domain) of Bax and one peptide binds to the outside of the BH3 domain (2-helix). Notably, the peptides restrict the flexibility of the 1-2 loop, modulating the bottom trigger site associated with toxicity. All in all, the results highlight the potential of these peptides as valuable tools for further exploration in modulating apoptotic pathways and set the structural foundation for a machine learning powered engine for peptide design.

bioengineering↗

bioRxiv2023

Structural adaptability and surface activity oftardigrade-inspired peptides

Tardigrades are unique micro-animals that withstand harsh conditions, such as extreme temperatures and desiccation. Recently, it was found that specific cytoprotective proteins are essential for ensuring this high environmental tolerance. In particular, cytoplasmic abundant heat soluble (CAHS) proteins, which are intrinsically disordered, adopt more ordered conformations upon desiccation, and are involved in the vitrification of the cytoplasm. The design and synthesis short peptides capable of mimicking the structural behavior (and thus the cytoprotective properties) of CAHS proteins would be beneficial for potential biomedical applications, including the development of novel heat-resistant preservatives for sensitive drug formulations. As a first step in this direction, we selected several model peptides of varying lengths derived from the conserved CAHS motifs 1 and 2, which are part of the intrinsically disordered CAHS-c2 region. We then studied their structures using circular dichroism and linear and two-dimensional infrared spectroscopy in the presence of the desolvating agent TFE (2,2,2-trifluoroethanol), which mimics desiccation. We found that the CAHS model peptides are mostly disordered at 0% TFE (a result that we confirmed by molecular dynamics simulations), but adopt a more -helical structure upon the addition of the desolvating agent, similar to what is observed for full CAHS proteins. Additionally, we employed sum frequency generation to investigate the surface activity of the peptides at the air/water interface to mimic a partial dehydration effect. Interestingly, all model peptides are surface active and also adopt a helical structure at the air/water interface. Thus, the selected sequences represent promising model peptides that show similarities in the physicochemical behavior to full CAHS proteins. Our results also suggest that arginine might be a crucial element in defining the strong propensity of these peptides to adopt a helical structure. In the future, the use CAHS model peptides to design new synthetic peptide-based materials could make it possible to mimic and exploit the cytoprotective properties of naturally occurring tardigrade proteins. SIGNIFICANCETardigrades are micro-animals that can survive extreme conditions such as desiccation and high temperatures. Recent work has shown that this capability is related to the presence of specific proteins that can remodel in order to protect the organisms cells. Mimicking this behavior using small peptides that preserve the structural properties of the full proteins is highly desirable in potential biomedical applications, such as the storage of heat-sensitive drugs. Here, we study the structural properties of model peptides derived from the conserved region of cytoplastic tardigrade proteins, and show that these peptides preserve some of the conformational behavior of the full protein under drying conditions. These peptides can therefore be used as a starting point for the design of synthetic model systems based on tardigrade-inspired peptides for tailored applications.

biochemistry↗

bioRxiv2023

Elucidating the role of water in collagen self assembly by isotopically modulating collagen hydration

Water is known to play an important role in collagen self assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self assembly. We dissolve collagen in H2O and D2O, and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D2O than in H2O, and collagen in D2O self assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions, which appear to be crucial in determining the acceleration of the initial nucleation rate. These results show that water acts as a mediator between collagen monomers, by moderating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self assembly.

biophysics↗

bioRxiv2021

The role of the first transmembrane helix in bacterial YidC: Insights from the crystal structure and molecular dynamics simulations

The evolutionary conserved YidC is a unique dual-function protein that adopts insertase and chaperone conformations. The TM1 helix of Escherichia coli YidC mediates the interaction between the YidC chaperone and the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the TM1 helix (PDB ID: 6Y86). The TM1 helix lies on the periplasmic side of the bilayer forming an angle of about 15 degrees with the membrane surface. Our functional studies suggest a role of TM1 for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that the TM1 helix acts as a membrane anchor for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of the TM1 helix and the other structural elements.

biophysics↗

bioRxiv2021

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Designed Ankyrin Repeat Proteins (DARPins) are a class of antibody mimetics with a high and mostly unexplored potential in drug development. They are clinically validated and thus represent a true alternative to classical immunoglobulin formats. In contrast to immunoglobulins, they are built from solenoid protein domains comprising an N-terminal capping repeat, one or more internal repeats and a C-terminal capping repeat. By using in silico analysis and a rationally guided Ala-Scan, we identified position 17 of the N-terminal capping repeat to play a key role for the overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by about 8{degrees}C to 10{degrees}C when the original Asp17 was replaced by Leu, Val, Ile, Met, Ala or Thr, as shown by high-temperature unfolding experiments at equilibrium. We then transferred the Asp17Leu mutation to various backgrounds, including different N- and C-terminal capping repeats and clinically validated DARPin domains, such as the VEGF-binding ankyrin repeat domain of abicipar pegol. In all cases, the proteins remained monomeric and showed improvements in the thermostability of about 8{degrees}C to 16{degrees}C. Thus, the replacement of Asp17 seems to be generically applicable to this drug class. Molecular dynamics simulations show that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, such a beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development. This mutation is likely responsible, at least in part, for the very high melting temperature (>90{degrees}C) of this promising anti-Covid-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.

biochemistry↗

bioRxiv2020

Flanking sequence preference modulates de novo DNA methylation in the mouse genome

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences are not mediated by the differential recruitment of the two de novo DNMTs to the genome but are resulting from structural differences in their catalytic domains. Molecular dynamics simulations suggest that, in case of DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. This context-dependent de novo DNA methylation provides additional insights into the complex regulation of methylation patterns in different cell types.

molecular biology↗