Biology subjects

Ries, F.

Publications and source records attributed to Ries, F..

5 recordsLinked to original sources

bioRxiv2025

Characterization of the TREM-1 Signaling Landscape in Human Neutrophils

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily, and an activating receptor mainly expressed on myeloid cells. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor-associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel targets for treating inflammatory and cancer diseases. As many specific kinase inhibitors are approved for treating various diseases, targeting kinases being active after TREM-1 ligation serves as a promising approach. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) by kinome activity profiling of purified human neutrophils after TREM-1 activation. As TREM-1 interacts with Toll-like receptor (TLR) 4 signaling, we used TLR4-activation by LPS to define TREM-1 specific pathways. We found an increased kinome activity after receptor ligation and could predict individual kinases. To gain further insights into the signaling cascade, we additionally investigated the transcriptomic profile that made us available to link the kinome activity to the resulting transcriptomic profile. In sum, we revealed several signaling pathways being active after TREM-1 ligation that are associated with various biological processed and diseases. This study facilitates selecting kinase inhibitors for further validation with the aim of targeting TREM-1 signaling in various inflammatory or cancer disease conditions.

immunology↗

bioRxiv2024

Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

synthetic biology↗

bioRxiv2023

Networked proteins redundantly interact with VAP27 and RABG3 to regulate membrane tethering at the vacuole and beyond

Biological processes in eukaryotes depend on the spatio-temporal compartmentalization of their cells. Integrity and positioning of organelles on the other hand rely on the organization of the actin cytoskeleton. Previously, it has been shown that changes of the plants largest organelle, the vacuole, depend on a functional actin organization. The connection between actin filaments and the vacuole is established by the family of Networked (NET) 4 proteins and, consequently, altering NET4 abundance impacts vacuolar morphology. However, the precise regulatory mechanism is unknown and gene deletions of NET4 did not result in a global growth phenotype. Here, we show that NET4 functions redundantly with NET3, interacting with RABG3-GTPases at the vacuole to allow for homotypic fusion or, alternatively, the generation of endoplasmic reticulum (ER) - vacuole contact sites. We found that ER-resident NET3 is able to interact with RABG3 residing at the tonoplast and that NET4 interacts with the contact site protein VAP27-1 at the ER. Generation of net3 net4 triple mutants by CRSIPR-guided mutagenesis helped us to overcome functional redundancy, resulting in impaired plant growth and development. Our results demonstrate how diversification of NET genes led to functional redundancy between different family members to create cellular plasticity of vascular plants. We hypothesize that establishment of a direct ER-vacuole connection enables direct lipid and protein transfer which is especially important in young and fast-growing cells. Availability of lipids would facilitate rapidly expanding vacuoles, which are the basis for high cell elongation rates and eventually fast plant growth.

cell biology↗

bioRxiv2023

Competition co-immunoprecipitation reveals interactors of the chloroplast CPN60 chaperonin machinery

The functionality of essential metabolic processes in chloroplasts depends on a balanced integration of nuclear-and chloroplast-encoded polypeptides into the plastids proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin of Escherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of a very limited knowledge about the folded substrates. We employed the competition immunoprecipitation method for the identification of the chaperonins substrates in Chlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL, we revealed numerous new substrates with high confidence. Identified substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical content and increased aggregation propensity. Immunoprecipitations performed with a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, with altered interactions during heat stress, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

molecular biology↗

bioRxiv2019

The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network

Chloroplast gene expression is tightly regulated and majorly controlled on the level of protein synthesis. Fine-tuning of translation is vital for plant development, acclimation to environmental challenges and for the assembly of major protein complexes such as the photosynthesis machinery. However, many regulatory mediators and the interaction network of chloroplast ribosomes are not known to date. We report here on a deep proteomic analysis of the plastidic ribosome interaction network in Chlamydomonas reinhardtii cells. Affinity-purification of ribosomes was achieved via endogenous affinity tagging of the chloroplast-encoded protein Rpl5, yielding a specific enrichment of >650 chloroplast-localized proteins. The ribosome interaction network was validated for several proteins and provides a new source of mainly conserved factors directly linking translation with central processes such as protein folding, photosystem biogenesis, redox control, RNA maturation, energy and metabolite homeostasis. Our approach provided the first evidence for the existence of a plastidic co-translational acting N-acetyltransferase (cpNAT1). Expression of tagged cpNAT1 confirmed its ribosome-association, and we demonstrated the ability of cpNAT1 to acetylate substrate proteins at their N-terminus. Our dataset establishes that the chloroplast protein synthesis machinery acts as nexus in a highly choreographed, spatially interconnected protein network and underscores its wide-ranging regulatory potential during gene expression. ONE-SENTENCE SUMMARYAffinity purification of Chlamydomonas reinhardtii chloroplast ribosomes and subsequent proteomic analysis revealed a broad spectrum of interactors ranging from global translation control to specific pathways.

biochemistry↗