Biology subjects

Bauer, N.

Publications and source records attributed to Bauer, N..

7 recordsLinked to original sources

bioRxiv2026

Dynamic Patterns of Nuclear Transcription Factor Abundance in Plant Basal Immunity Revealed by Spatial Proteomics of Arabidopsis Nuclei

O_LIThe control of amount of nuclear proteins is fundamental in regulating plant gene expression, but the mechanisms of quantitative dynamics of the nuclear proteome are largely unstudied during adaptive responses to pathogens. C_LIO_LIHighly specific labeling, enrichment and measurement of the nuclear proteome was performed using TurboID LC-MS of Arabidopsis thaliana leaves treated with the pathogen-associated molecular pattern (PAMP), flg22, and/or cycloheximide. The chosen experimental approach allowed discrimination of the effects of translation, nuclear protein import, trafficking of preexisting proteins, derepression, and nuclear protein turn-over upon elicitation of basal immunity. C_LIO_LIThe highly specific, deep coverage of proteins in the nucleus makes this study a resource for anyone interested in plant nuclear proteome dynamics and defense. C_LIO_LIAround 2,000 nuclear proteins were repeatedly quantified, including more than 300 transcription factors or other proteins related to transcription. Several proteins with documented activity in endosomes were newly synthesized and imported into nuclei upon PAMP challenge, suggesting alternative nuclear functions in PAMP-triggered immunity (PTI). Circadian clock components, including the transcription factor, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-HIKING EXPEDITION (CHE), were depleted upon PAMP challenge, suggesting a safeguard against untimely induction of systemic acquired resistance (SAR). C_LIO_LIBased on proteomic patterns, proteins moonlighting in the nucleus as well as trafficking and turn-over regulation of the proteome are common elements during plant immunity. C_LI

plant biology↗

bioRxiv2025

Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody crosslinking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies (mAbs) daratumumab and isatuximab, and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies. One-Sentence SummarySingle-molecule localization microcopy quantifies the expression and resolves the stoichiometry of endogenous membrane receptors

biophysics↗

bioRxiv2024

The endophytic fungus Serendipita indica alters auxin distribution in Arabidopsis thaliana roots through alteration of auxin transport and conjugation to promote plant growth

Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of inter-organismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Co-colonization of Arabidopsis roots with S. indica significantly promotes plant growth. Due to the notable phenotypic alterations of fungus-infected root systems, the involvement of a reprogramming of plant hormone levels, especially that of indole-3-acetic acid, has been suggested earlier. However, until now, the molecular mechanism by which S. indica promotes plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica. Our experiments revealed the essential role of tightly controlled auxin conjugation in the plant-fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 (GH3) genes, GH3.5 and GH3.17, for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signaling in roots, which results in increased plant growth.

plant biology↗

bioRxiv2023

Targeting LC3/GABARAP for degrader development and autophagy modulation

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP by in silico docking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

biochemistry↗

bioRxiv2023

Development of potent dual BET/HDAC inhibitors via pharmacophore merging and structure-guided optimization

Bromodomain and extra-terminal motif (BET) proteins and histone deacetylases (HDACs) are prime targets in cancer therapy. Recent research has particularly focused on the development of dual BET/HDAC inhibitors for hard-to-treat tumors such as pancreatic cancer. Here, we have developed a new series of potent dual BET/HDAC inhibitors by choosing starting scaffolds that enabled us to optimally merge the two functionalities into a single compound. Systematic structure-guided modification of both warheads then led to optimized binders that were superior in potency to both parent compounds, with the best molecules of this series binding to both BRD4 bromodomains as well as HDAC1/2 with EC50 values in the 100-nanomolar range in cellular NanoBRET target engagement assays. Importantly, this on-target activity also translated into promising efficacy in pancreatic cancer and NUT midline carcinoma cells. Our lead molecules effectively blocked histone H3 deacetylation in pancreatic cancer cells and upregulated the tumor suppressor HEXIM1 and proapoptotic p57, both markers of BET inhibition. In addition, they have the potential to downregulate oncogenic drivers of NUT midline carcinoma, as demonstrated for MYC and TP63 mRNA levels. Overall, this study expands the portfolio of available dual BET/class I HDAC inhibitors for future translational studies in different cancer models.

biochemistry↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2019

Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.\n\nSynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

immunology↗