Biology subjects

Rotsides, P.

Publications and source records attributed to Rotsides, P..

4 recordsLinked to original sources

bioRxiv2026

A generalizable system for antigenic peptide targeting across HLA-I allotypes

T cell receptors (TCRs) and TCR-mimicking antibodies recognize peptide antigens in the context of specific Human Leucocyte Antigen (HLA-I) allotypes, and the extreme polymorphism of the HLA locus limits the breadth of immunotherapy development. Key barriers include divergent molecular surfaces on HLA proteins and differences in the peptide structure. As a result, existing modalities cannot confer therapeutic coverage across patients of divergent genetic backgrounds. Here, we develop an approach which combines a peptide conformational prediction tool, PepPred, with a cross-HLA binding protein engineering system, TRACeR-I1, to outline a generalized framework for developing binders (xTRACeRs) with compatibility across HLA allotypes while maintaining high levels of specificity towards the peptide antigen. We use our system to develop and validate xTRACeRs against clinically relevant, established peptide antigens presented across common alleles within five HLA-A/B/C supertypes2. Cryo-EM structures of xTRACeR-pHLA complexes for an oncofetal antigen from PRAME and a neuroblastoma-specific peptide from PHOX2B reveal effective mechanisms to navigate polymorphic HLA surface residues, and extensive interactions with the peptide. We implemented these two xTRACeRs as Chimeric Antigen Receptor (CAR) T cells and demonstrated their potent killing efficacy and specificity. Overcoming restriction across HLA supertypes lifts a key barrier in HLA-targeted immunotherapy by expanding patient coverage.

synthetic biology↗

bioRxiv2026

A miniaturized MR1 metabolite display system with native-like protein features

Major histocompatibility complex class I-related protein 1 (MR1) presents metabolite-derived antigens to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells, playing a critical role in immune surveillance during infection and disease. Biochemical and structural studies of MR1 have been limited by the intrinsic instability of the molecule, which requires both ligand binding and association with beta-2-microglobulin ({beta}2m) for proper folding and stability. Here, we adapt MR1 to the SMART protein platform to generate a minimalistic system for studying MR1 ligand presentation and T cell receptor (TCR) recognition. SMART-MR1 consists of the MR1 1/2 ligand-binding platform fused to a helical stabilizing domain that functionally replaces the 3 and {beta}2m domains, resulting in a truncated protein that preserves the architecture of the antigen-binding groove. We show that SMART-MR1 can be efficiently produced recombinantly and retains the ability to bind chemically diverse classes of MR1 ligands. The reduced size of SMART-MR1 enables amide-based solution NMR experiments, and its simplified structure allows for ligand screening using fluorescence polarization. Importantly, SMART-MR1 maintains binding to the MAIT-derived A-F7 TCR, as confirmed by isothermal titration calorimetry. Finally, cryo-EM structural analysis of SMART-MR1/5-OP-RU bound to A-F7 revealed that ligand presentation and TCR recognition are nearly identical to those observed in native MR1. Together, these results establish SMART-MR1 as a minimal yet native-like system, expanding the experimental toolkit available for studying MR1 interactions and facilitating future efforts aimed at targeting MR1 pathways. Significance StatementMR1 is a highly conserved antigen-presenting molecule that enables T cells to detect metabolite signals from microbial infections and host metabolism. Despite its importance in immunity, mechanistic studies of MR1 have been limited by the instability of the native protein. We developed a simplified and stabilized version of MR1 that preserves the ligand-binding platform while eliminating structural elements that complicate biochemical analysis. This minimal system retains native-like antigen presentation and T cell receptor (TCR) recognition, while enabling experimental approaches that are difficult with full-length MR1. By lowering technical barriers to studying MR1-ligand and MR1-TCR interactions, this platform provides a versatile tool for exploring how antigens shape immune responses and for accelerating discovery of therapeutic strategies targeting MR1.

biochemistry↗

bioRxiv2023

The VanS sensor histidine kinase from type-B VRE recognizes vancomycin directly

Vancomycin-resistant enterococci (VRE) are among the most common causes of nosocomial infections and have been prioritized as targets for new therapeutic development. Many genetically distinct types of VRE have been identified; however, they all share a common suite of resistance genes that function together to confer resistance to vancomycin. Expression of the resistance phenotype is controlled by the VanRS two-component system. This system senses the presence of the antibiotic, and responds by initiating transcription of resistance genes. VanS is a transmembrane sensor histidine kinase, and plays a fundamental role in antibiotic resistance by detecting vancomycin or its effects; it then transduces this signal to the VanR transcription factor, thereby alerting the organism to the presence of the antibiotic. Despite the critical role played by VanS, fundamental questions remain about its function, and in particular about how it senses vancomycin. Here, we focus on a purified VanRS system from one of the most clinically prevalent forms of VRE, type B. We show that in a native-like membrane environment, the autokinase activity of type-B VanS is strongly stimulated by vancomycin. We additionally demonstrate that this effect is mediated by a direct physical interaction between the antibiotic and the type-B VanS protein, and localize the interacting region to the proteins periplasmic domain. This represents the first time that a direct sensing mechanism has been confirmed for any VanS protein. Significance StatementWhen vancomycin-resistant enterococci (VRE) sense the presence of vancomycin, they remodel their cell walls to block antibiotic binding. This resistance phenotype is controlled by the VanS protein, a histidine kinase that senses the antibiotic or its effects and signals for transcription of resistance genes. However, the mechanism by which VanS detects the antibiotic has remained unclear, with no consensus emerging as to whether the protein interacts directly with vancomycin, or instead detects some downstream consequence of vancomycins action. Here, we show that for one of the most clinically relevant types of VRE, type B, VanS is activated by direct binding of the antibiotic. Such mechanistic insights will likely prove useful in circumventing vancomycin resistance.

biochemistry↗

bioRxiv2023

Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Vancomycins interactions with cellular targets drive its antimicrobial activity, and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycins interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications, as compared to previous photoprobes. Using proteins fused to vancomycins main cell-wall target, D-alanyl-D-alanine, we use mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying novel vancomycin-binding proteins. For Table of Contents Only. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/545140v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d21b8forg.highwire.dtl.DTLVardef@10f4d4borg.highwire.dtl.DTLVardef@19bb1f0org.highwire.dtl.DTLVardef@bf3dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗