Biology subjects

Mergner, J.

Publications and source records attributed to Mergner, J..

10 recordsLinked to original sources

bioRxiv2025

OVEREXPRESSION OF TUMORPROTECTIVE CLUSTERIN PREVENTS EXHAUSTION OF TRANSGENIC T CELLS

1T cell therapies, such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) transgenic (tg) T cells, are a promising approach in the treatment of solid malignancies but are limited by T cell exhaustion caused by chronic antigen stimulation. Clusterin (CLU) is a chaperone protein known to protect both normal and malignant cells, from metabolic stress and reactive oxygen species (ROS). In this study, we investigated whether overexpression (OE) of CLU in TCRtg T cells and CAR-T cells respectively can reduce exhaustion induced by chronic antigen stimulation and enhance T cell functionality against Ewing sarcoma (EwS). Among other cytoprotective genes, we found that CLU was significantly downregulated in dysfunctional tumor infiltrating lymphocytes. Therefore, we engineered EwS-directed TCRtg T cells targeting a Chondromodulin-1 (Chm1)-derived peptide and GD2 CAR-T cells to overexpress CLU. We show here that CLU is downregulated in T cells following activation by tumor cells. CLU-overexpressing T cells exhibit decreased expression of exhaustion markers (PD1, LAG3) and reduced apoptosis after repetitive stimulation. These cells demonstrated improved infiltration into tumor spheroids and maintained functionality under hypoxic conditions. In vivo, CLU-overexpressing T cells showed enhanced persistence and a trend towards reduced tumor growth. Mechanistically, proteomic analysis suggested that reduced ribosomal activity might delay T cell exhaustion, implicating metabolic reprogramming. In conclusion, CLU OE in tg T cells enhances their persistence and functionality by mitigating exhaustion, possibly through modulation of ribosomal activity and metabolic pathways. This strategy holds potential for improving adoptive T cell therapies against solid tumors.

immunology↗

bioRxiv2025

Apoptotic caspases silence spontaneous innate immune signals by specifically cleaving activated mitochondrial antiviral signalling protein (MAVS)

Caspases-9, -3 and -7 are activated in the mitochondrial apoptosis pathway and lead to the apoptotic phenotype. Caspases also function to limit inflammation upon apoptotic mitochondrial permeabilization through degradation of the signalling proteins cGAS, MAVS and IRF3. Cells and mice lacking caspases have higher interferon levels and are resistant to viral infection. We report that in unstimulated, non-apoptotic cells caspase-3 functions to cleave specifically activated MAVS and very likely cGAS. In unstimulated HeLa cells, constitutive caspase-9- and -3-but not 7-dependent proteolytic events were observed. Inhibition of the mitochondrial apoptosis pathway in various healthy cells induced type I interferon (IFN I) through increased cGAS activity in the absence of changes to cGAS levels. We observed enhanced MAVS-dependent signals upon RIG-I-like helicase stimulation in the absence of BAX, caspase-9 or caspase-3 or upon caspase-inhibition. During activation, MAVS forms complexes, and blockade of mitochondrial apoptosis signalling increased complex abundance in unstimulated and stimulated cells. MAVS complexes were more sensitive to caspase-degradation than the monomer, and mutation of caspase-3-cleavage sites in MAVS spontaneously increased complex formation. Inhibition of voltage-dependent anion channel 1 (VDAC1) oligomerization blocked BAX/BAK- and caspase-regulated IFN induction, suggesting a stimulating role of leakage of mitochondrial DNA. We propose that low level, spontaneous activity of the mitochondrial apoptosis pathway, through specific caspase-3-mediated cleavage of only active signaling proteins, counteracts mitochondrial release of nucleic acids to reduce inflammation in the absence of infection. Caspase-3 therefore has a novel function in conformation- and activation-specific cleavage of substrates.

cell biology↗

bioRxiv2025

Structural Basis for OAS2 Regulation and its Antiviral Function

Oligoadenylate synthetase (OAS) proteins are immune sensors for double-stranded RNA and critical for restricting viruses. OAS2 comprises two OAS domains, only one of which can synthesize 2-5-oligoadenylates for RNase L activation. Existing structures of OAS1 provide a model for enzyme activation, but do not explain how multiple OAS domains discriminate RNA length. Here, we discover that OAS2 exists in an autoinhibited state as a zinc-mediated dimer and present a mechanism for RNA length discrimination: the catalytically deficient domain acts as a molecular ruler that prevents autoreactivity to short RNAs. We demonstrate that dimerization and myristoylation localize OAS2 to Golgi membranes and that this is required for OAS2 activation and restriction of viruses that exploit the endomembrane system for replication, e.g. coronaviruses. Finally, our results highlight the non-redundant role of OAS proteins and emphasize the clinical relevance of OAS2 by identifying a patient with a loss-of-function mutation leading to autoimmune disease.

biochemistry↗

bioRxiv2024

Barley resistance and susceptibility to fungal cell entry involve the interplay of ROP signaling with phosphatidylinositol-monophosphates

Rho-of-plant small GTPases (ROPs) are regulators of plant polar growth and of plant-pathogen interactions. The barley ROP, RACB, is involved in susceptibility towards infection by the barley powdery mildew fungus Blumeria hordei (Bh) but little is known about the cellular pathways that connect RACB-signaling to disease susceptibility. Here we identify novel RACB interaction partners of plant or fungal origin by untargeted co-immunoprecipitation of constitutively active (CA) RACB tagged by green-fluorescent protein from Bh-infected barley epidermal layers and subsequent analysis by liquid chromatography-coupled mass spectrometry. Three of immunoprecipitated proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase and a putative Bh-effector protein are involved in the barley-Bh-pathosystem and support disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in case of RACB, this lipid-interaction is mediated by its carboxy-terminal polybasic region (PBR). Fluorescent markers for anionic phospholipids show altered subcellular distribution in barley cells during Bh-attack and under expression of a RACB-binding fungal effector. Phosphatidylinositol 4-phosphate, phosphatidylinositol 3,5-bisphosphate and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. The interplay of ROPs with anionic phospholipids and phospholipid-metabolizing enzymes may thus enable the subcellular enrichment of components pivotal for success or failure of fungal penetration.

plant biology↗

bioRxiv2024

Transport properties of canonical PIN-FORMED proteins and the role of the loop domain in auxin transport

Indole-3-acetic acid (IAA), the most abundant endogenous auxin is transported in plants in a polar fashion by PIN-FORMED (PIN) transporters and controls virtually all plant growth and developmental processes. Canonical PINs possess a long and largely disordered cytosolic loop domain which is shorter in non-canonical PINs. Auxin transport by canonical PINs is activated loop phosphorylation by kinases. While the structure of the transmembrane domains of these transporters was recently solved, their transport properties remained poorly characterized and particularly the relative roles of the transmembrane and loop domain therein. In this study we used flux studies to obtain quantitative kinetic parameters of IAA transport mediated by canonical PINs as well as of chimeras between transmembrane and loop domains of different PINs upon their activation by D6 PROTEIN KINASE or PINOID. We found that the transporters possess distinct transport properties that are due to both the transmembrane and loop domain. To demonstrate the physiological relevance of these distinct transport properties, we modelled root tip IAA distribution patterns and investigated the potential of different PINs to complement the agravitropic root growth phenotype of the pin2 mutant when expressed in the PIN2 domain. We found a strong correlation between transport parameters and physiological output indicating that in addition to PIN polarity a low transport rate in the PIN2 expression domain is required for gravitropic growth. Overall, the data show that the loop domain is not only required for activation of PIN-mediated auxin transport but has an additional role in the transport cycle by a currently unknown mechanism.

plant biology↗

bioRxiv2023

ARID1A Mutations Protect Follicular Lymphoma from FAS-dependent Immune Surveillance by Reducing RUNX3/ETS1-Driven FAS-Expression

The cell death receptor FAS and its ligand (FASLG) play crucial roles in the selection of B cells during the germinal center (GC) reaction. Failure to eliminate potentially harmful B cells via FAS can lead to lymphoproliferation and the development B cell malignancies. The classic form of follicular lymphoma (FL) is a prototypic GC-derived B cell malignancy, characterized by the t(14;18) (q32;q21)IGH::BCL2 translocation and overexpression of antiapoptotic BCL2. Additional alterations were shown to be clinically relevant, including mutations in ARID1A. ARID1A is part of the SWI/SNF nucleosome remodeling complex that regulates DNA accessibility ("openness"). However, the mechanism how ARID1A mutations contribute to FL pathogenesis remains unclear. We analyzed 151 FL biopsies of patients with advanced stage disease at initial diagnosis and found that ARID1A mutations were recurrent and mainly disruptive, with an overall frequency of 18%. Additionally, we observed that ARID1A mutant FL showed significantly lower FAS protein expression in the FL tumor cell population. Functional experiments in BCL2-translocated lymphoma cells demonstrated that ARID1A is directly involved in the regulation of FAS, and ARID1A loss leads to decreased FAS protein and gene expression. However, ARID1A loss did not affect FAS promotor openness. Instead, we identified and experimentally validated a previously unknown co-transcriptional complex consisting of RUNX3 and ETS1 that regulates FAS expression, and ARID1A loss leads to reduced RUNX3 promotor openness and gene expression. The reduced FAS levels induced by ARID1A loss rendered lymphoma cells resistant to both soluble and T cell membrane-anchored FASLG-induced apoptosis. In summary, we have identified a functionally and clinically relevant mechanism how FL cells can escape FAS-dependent immune surveillance, which may also impact the efficacy of T cell-based therapies, including bispecific antibodies and CAR T cells.

cancer biology↗

bioRxiv2023

NF-κB c-REL-OTUD4 axis regulates B-cell receptor in B-cell lymphoma

The B-cell receptor (BCR) is essential for B-cell development and a crucial clinical target in immuno-oncology. However, therapeutic success against the BCR and downstream signaling pathways is hampered by enhanced NF-{kappa}B activation as a resistance mechanism. Using a multiomic approach, we discover the c-REL proto-oncogenic subunit of the NF-{kappa}B family as a key transcription factor regulating BCR subunit levels in B-cell lymphoma. Subsequent ChIP- seq, cell biology experiments, and patient data analysis reveal that OTUD4 is a critical deubiquitinase for inhibiting proteasomal degradation of c-REL and for stabilizing a multi-loop positive feedback of NF-{kappa}B to the BCR pathway. Remarkably, OTUD4 downregulation destabilizes c-REL and BCR levels and inhibits cell growth of B cell lymphoma. Thus, we shed light on the malignant potential of c-REL abundance, identify a positive feedback from c-REL to upstream BCR and present OTUD4 as a vulnerability to synergistically target NF-{kappa}B and BCR pathways in B-cell lymphoid malignancies.

cancer biology↗

bioRxiv2023

The RALF Signaling Pathway Regulates Cell Wall Integrity during Pollen Tube Growth in Maize

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis. To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize. We show that Clade IB ZmRALF2/3 mutants, but not that of Clade III ZmRALF1/5 caused cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located to the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution pattern leading to altered cell wall thickness, hyperphosphorylation of ZmPEX cell wall proteins and pollen tube burst. Clade IB, but not Clade III ZmRALFs are capable to interact with pollen-specific CrRLK1L receptor kinases ZmFERL4/7/9 and GPI-anchored co-receptors ZmLLG1/2 at similar binding affinities. In contrast, binding affinity to ZmPEX2/4 cell wall proteins is about five times higher. Based on these data, we now propose a dosage-dependent model showing how Clade IB RALFs act as extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in plants. One sentence summaryPollen-specific RALFs interact at different binding affinities with receptor kinases, GPI-anchored proteins and cell wall proteins to regulate cell wall integrity during pollen tube growth in maize.

developmental biology↗

bioRxiv2021

A hetero-oligomeric remorin-receptor complex regulates plant development

Plant growth and development are modulated by both biotic and abiotic stress. Increasing evidence suggests that cellular integration of the corresponding signals occurs within preformed hubs at the plasma membrane called nanodomains. These membrane sub-compartments are organized by multivalent molecular scaffold proteins, such as remorins. Here, we demonstrate that Group 1 remorins form a hetero-oligomeric complex at the plasma membrane. While these remorins are functionally redundant for some pathways their multivalency also allows the recruitment of specific interaction partners. One of them, the receptor-like kinase REMORIN-INTERACTING RECEPTOR 1 (RIR1), that acts redundantly with the closely related receptor NILR2, is specifically recruited by REM1.2 in a phosphorylation-dependent manner. Overlapping developmental phenotypes suggest that the REM/RIR complex regulates key developmental pathways.

plant biology↗

bioRxiv2020

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

plant biology↗