Biology subjects

Stephan, P.

Publications and source records attributed to Stephan, P..

4 recordsLinked to original sources

bioRxiv2024

Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

cancer biology↗

bioRxiv2023

Directed evolution of piperazic acid incorporation by a nonribosomal peptide synthetase

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are non-ribosomal peptide synthetases (NRPSs) responsible for production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by LC-MS/MS based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analog. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

synthetic biology↗

bioRxiv2023

Screening megasynthetase mutants at high throughput using droplet microfluidics

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases the number of stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

bioengineering↗

bioRxiv2020

Engineering DNA templated nonribosomal peptide synthesis

Nanocontainers or macromolecular scaffolds for artificial biocatalytic cascades facilitate sequential enzyme reactions but diffusive escape of intermediates limits rate enhancement. Nonribosomal peptide synthetases (NRPS) naturally form gigantic assembly lines and prevent escape by covalently tethering intermediates. Here, we have built DNA-templated NRPS (DT-NRPS) by adding zinc finger tags to split NRPS modules. The zinc fingers direct the NRPS modules to 9-bp binding sites on a DNA strand, where they form a catalytically active enzyme cascade. DT-NRPS outperform previously reported DNA templated enzyme cascades in terms of DNA acceleration which demonstrates that covalent intermediate channeling is possible along the DNA template. Attachment of assembly line enzymes to a DNA scaffold is a promising catalytic strategy for the sequence-controlled biosynthesis of nonribosomal peptides and other polymers.

synthetic biology↗