Biology subjects

Alici, E.

Publications and source records attributed to Alici, E..

6 recordsLinked to original sources

bioRxiv2025

From Structure to Immunogenicity: Decoding Correlated Dynamics atthe Peptide MHC interface to Understand TCR Recognition

The interaction between a class I peptide-major histocompatibility complex (pMHC) and a T cell receptor (TCR) plays a central role in the elicitation of CD8+ T cell immune responses. As a result, considerable effort has been invested in understanding the structural, dynamic, and biophysical parameters that govern this recognition event, including designing altered peptide ligands (APLs) which seek to modulate the downstream signaling outcomes. However, dynamic links between modified peptide positions and distant residues have until yet been ill resolved. Using an integrative approach combining crystallographic ensemble and single models with atomistic molecular dynamics simulations and correlational analysis, we have established an approach that allows us to identify coupled dynamics between spatially distant residues at the pMHC interface. Furthermore, we constructed a network encoding the inter-residue couplings observed throughout the simulations. This computational workflow corroborates well with experimental data and leads to novel insights regarding the differential immunogenicity of the closely related peptides analyzed in this study. Ultimately, we present an intuitive and comprehensive strategy for decoding the linked dynamics at the pMHC interface allowing for mechanistic insights into the biophysical bases governing immunogenicity. One Sentence SummaryThe dynamics at the pMHC interface can be encoded as a biophysically relevant network to yield molecular insights into immunogenicity

immunology↗

bioRxiv2025

SAXS reveals the molecular basis underlying pH-driven G3BP1 conformational dynamics: implications for stress granule formation

G3BP1 is the central node and molecular switch in stress granule (SG) assembly. However, structural insights into full-length G3BP1 remain elusive owing to its extensive intrinsically disordered regions (IDRs). Using size-exclusion chromatography-coupled small-angle X-ray scattering (SEC-SAXS), we have characterized the solution architecture and conformational dynamics of full-length G3BP1. Under physiological conditions, G3BP1 adopts an elongated, head-to-head antiparallel homodimeric conformation, whereas acidification induces a pronounced conformational compaction. Subsequent biophysical studies reveal that this compact state enables robust RNA-mediated and, notably, homotypic phase separation in vitro. Deletion of the RGG region abolishes this acidity-induced compaction and markedly impairs phase separation, establishing a causal link between the RGG-dependent conformational switch and phase separation propensity. By moving beyond hypothetical models to experimental solution-state data, our work fills a longstanding void in the field and provides critical insights into the structural plasticity that underlies G3BP1 function, offering a missing structural link essential for deciphering the molecular mechanism of SG formation. We propose that stress-associated physicochemical changes, specifically localized acidification coupled with mRNA accumulation, trigger this reversible structural reconfiguration of G3BP1, thereby facilitating phase separation.

molecular biology↗

bioRxiv2025

Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

The compromise between affinity and specificity in TCR-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge in developing efficacious immunotherapies. As such, T cell engagers which circumvent these limitations are of particular interest. We have established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I molecules in complex with tumor-associated peptides and CD3{varepsilon}. High-affinity HLA-A*0201/NY-ESO1157-165-specific DARPins were isolated after only four rounds of in-vitro selection from naive DARPin libraries. Combining HLA-A*0201/NY-ESO1157-165-specific DARPins with a CD3{varepsilon}-specific DARPin created potent T cell engagers which elicited CD8+ T cell activation towards tumor targets with high peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. The cryo-EM structure of a ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid and concave DARPin surface that binds to the entire length of the peptide-binding cleft, contacting both -helices and the peptide. The present results unveil promising immuno-oncotherapeutic approaches with the possibility of rapidly developing DARPins with high affinity and specificity to HLA/peptide targets that can be readily combined with a new generation of anti-CD3{varepsilon}-specific DARPins.

synthetic biology↗

bioRxiv2024

Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a significant challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the systems versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents a significant advancement in the feasibility and scalability of gene-editing delivery technologies.

bioengineering↗

bioRxiv2024

Crystallographic and NMR studies of Streptococcus pneumonia LCP protein PsrSp indicate the importance of dynamics in four long loops for ligand specificity

The crystal structure of the extracellular region of the second pneumococcal LCP, a polyisoprenyl-teichoic acid-peptidoglycan teichoic acid transferase PsrSp, was determined and refined to 2.15[A] resolution. Despite the low sequence homology with other LCP proteins, the PsrSp maintains the fold of the LCP domain and the positions of the 15 residues suggested to participate in the transferase function are conserved. The empty tunnel found in the PsrSp between the central {beta}-sheet and three -helices is wide enough to accommodate polyisoprenyl-teichoic acid. Comparison of the crystallographic temperature factors of LCP from distinct bacteria demonstrated that the four long loops located close to the teichoic acid and peptidoglycan binding sites have different relative mobility. To compare the dynamics of the PsrSp in crystalline state and in solution, NMR spectra were recorded, and 88% of the residues were assigned in the 1H-15N TROSY HSQC spectra. Comparison of the secondary structure of the crystal structure of PsrSp with NMR data demonstrated a perfect concordance between the results using these two methods. Moreover, the relative mobility of the essential loops estimated from the crystallographic B-factor is in good agreement with order parameter S2, predicted from chemical shift. We hypothesize that the dynamics of these loops are important for the substrate promiscuity of LCP proteins.

microbiology↗

bioRxiv2022

Control of NK cell tolerance in MHC class I-deficiency by regulated SHP-1 localization to the activating immune synapse

Signaling via inhibitory KIR/Ly49 receptors preserves natural killer (NK) cell self-tolerance but also conveys NK cell reactivity towards MHC class-I low target cells in an education process. Here, we demonstrate that mouse NK cell education by H-2Dd regulates transcription of several genes in Ly49A+ NK cells including Ptpn6, encoding the phosphatase SHP-1. SHP-1 was highly expressed in uneducated NK cells, in which knock-out of Ptpn6 increased responsiveness. Following NKp46 triggering of uneducated NK cells, a higher synaptic abundance of phosphorylated SHP-1 was found relative to educated NK cells, concomitant with reduced phosphorylation of several signaling molecules, including PLC-g2, SLP-76, ZAP70/Syk and ERK1/2. SHP-1 overlapped extensively with F-actin and SLP-76 in the uneducated activating synapse of Ly49A+ NK cells, whereas a greater association between Ly49A and SHP-1 was observed in educated NK cells. Thus, our results indicate that in addition to transcriptional regulation, a distinct SHP-1 patterning in NK cell activating synapses can determine their tolerance.

immunology↗